High performance fluorescence imaging module for genomic testing assay

ABSTRACT

Fluorescence imaging system designs are described that provide larger fields-of-view, increased spatial resolution, improved modulation transfer and image quality, higher spatial sampling frequency, faster transitions between image capture when repositioning the sample plane to capture a series of images (e.g., of different fields-of-view), and improved imaging system duty cycle, and thus enable higher throughput image acquisition and analysis for genomics and other imaging applications.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/372,361, filed Jul. 9, 2021, which is a continuation of InternationalApplication No. PCT/US2021/013696, filed Jan. 15, 2021, which claims thebenefit of U.S. Provisional Application No. 63/076,361, filed Sep. 9,2020, and U.S. Provisional Application No. 62/962,723, filed Jan. 17,2020, each of which is incorporated herein by reference in its entirety.

BACKGROUND

In typical fluorescence-based genomic testing assays, e.g., genotypingor nucleic acid sequencing (using either real time, cyclic, or stepwisereaction schemes), dye molecules that are attached to nucleic acidmolecules tethered on a substrate are excited using an excitation lightsource, a fluorescence photon signal is generated in one or morespatially-localized positions on the substrate, and the fluorescence issubsequently imaged through an optical system onto an image sensor. Ananalysis process is then used to analyze the images, find the positionsof labeled molecules (or clonally amplified clusters of molecules) onthe substrate, and quantify the fluorescence photon signal in terms ofwavelength and spatial coordinates, which may then be correlated withthe degree to which a specific chemical reaction, e.g., a hybridizationevent or base addition event, occurred in the specified locations on thesubstrate. Imaging-based methods provide large scale parallelism andmultiplexing capabilities, which help to drive down the cost andaccessibility of such technologies. However, detection errors that arisefrom, for example, overly dense packing of labeled molecules (orclonally-amplified clusters of molecules) within a small region of thesubstrate surface, or due to low contrast-to-noise ratio (CNR) in theimage, may lead to errors in attributing the fluorescence signal to thecorrect molecules (or clonally amplified clusters of molecules).

SUMMARY

Disclosed herein are imaging systems configured to image a firstinterior surface and a second interior surface of a flow cell, theimaging systems comprising: a) an objective lens; b) at least one imagesensor; and c) at least one tube lens disposed in an optical pathbetween the objective lens and the at least one image sensor; whereinsaid optical system has a numerical aperture (NA) of less than 0.6 and afield-of-view (FOV) of greater than 1.0 mm²; and wherein the at leastone tube lens is configured to correct imaging performance such thatimages of the first interior surface of the flow cell and the secondinterior surface of the flow cell have substantially the same opticalresolution.

In some embodiments, the flow cell has a wall thickness of at least 700μm and a fluid-filled gap between the first interior surface and thesecond interior surface of at least 50 μm. In some embodiments, theimages of the first interior surface and the second interior surface areacquired without moving an optical compensator into an optical pathbetween said objective lens and said at least one image sensor. In someembodiments, the imaging system has a numerical aperture (NA) of lessthan 0.6. In some embodiments, the imaging system has a numericalaperture (NA) of greater than 0.3. In some embodiments, the imagingsystem has a field-of-view (FOV) of greater than 1.5 mm². In someembodiments, the optical resolution of images of the first interiorsurface and the second interior surface are diffraction-limited acrossthe entire field-of-view (FOV). In some embodiments, the at least onetube lens comprises, in order, an asymmetric convex-convex lens, aconvex-plano lens, an asymmetric concave-concave lens, and an asymmetricconvex-concave lens. In some embodiments, the imaging system comprisestwo or more tube lenses which are designed to provide optimal imagingperformance for the first interior surface and the second interiorsurface at two or more fluorescence wavelengths. In some embodiments,the imaging system further comprises a focusing mechanism configured torefocus the optical system between acquiring images of the firstinterior surface and the second interior surface. In some embodiments,the imaging system is configured to image two or more fields-of-view onat least one of the first interior surface or the second interiorsurface. In some embodiments, the first interior surface and secondinterior surface of the flow cell are coated with a hydrophilic coatinglayer, and wherein said hydrophilic coating layer further compriseslabeled nucleic acid colonies disposed thereon at a surface densityof >10,000 nucleic acid colonies/mm². In some embodiments, an image ofthe first interior surface or second interior surface acquired using theimaging system shows a contrast to noise ratio (CNR) of at least 5 whenthe nucleic acid colonies are labeled with cyanine dye 3 (Cy3), theimaging system comprises a dichroic mirror and bandpass filter setoptimized for Cy3 emission, and the image is acquired under non-signalsaturating conditions while the surface is immersed in 25 mM ACES, pH7.4 buffer. In some embodiments, said imaging system comprises 1, 2, 3,or 4 imaging channels configured to detect nucleic acid coloniesdisposed on at least one of said two distinct surfaces that have beenlabeled with 1, 2, 3, or 4 distinct detectable labels. In someembodiments, the imaging system is used to monitor asequencing-by-avidity, sequencing-by-nucleotide base-pairing,sequencing-by-nucleotide binding, or sequencing-by-nucleotideincorporation reaction on at least one of the first interior surface andthe second interior surface and detect a bound or incorporatednucleotide base. In some embodiments, the imaging system is used toperform nucleic acid sequencing. In some embodiments, the imaging systemis used to determine a genotype of a sample, wherein determining thegenotype of the sample comprises preparing a nucleic acid moleculeextracted from the sample for sequencing, and then sequencing thenucleic acid molecule. In some embodiments, the at least one imagesensor comprises pixels having a pixel dimension chosen such that aspatial sampling frequency for the imaging system is at least twice anoptical resolution of the imaging system. In some embodiments, acombination of the objective lens and the at least one tube lens isconfigured to optimize a modulation transfer function in the spatialfrequency range from 700 cycles per mm to 1100 cycles per mm in thesample plane. In some embodiments, the at least one tube lens isdesigned to correct modulation transfer function (MTF) at one or morespecified spatial frequencies, defocus, spherical aberration, chromaticaberration, coma, astigmatism, field curvature, image distortion, imagecontrast-to-noise ratio (CNR), or any combination thereof, for acombination of the objective lens and the at least one tube lens.

Also disclosed herein are methods of sequencing a nucleic acid molecule,the methods comprising: a) imaging a first surface and anaxially-displaced second surface using an optical system which comprisesan objective lens and at least one image sensor, wherein said opticalsystem has a numerical aperture (NA) of less than 0.6 and afield-of-view (FOV) of greater than 1.0 mm², and wherein images of thefirst surface and the axially-displaced second surface havingsubstantially the same optical resolution are acquired without moving anoptical compensator into an optical path between said objective lens andsaid at least one image sensor; and b) detecting a fluorescently-labeledcomposition comprising the nucleic acid molecule, or a complementthereof, disposed on the first surface or the axially-displaced secondsurface to determine an identity of a nucleotide in the nucleic acidmolecule.

In some embodiments, a focusing mechanism is utilized to refocus theoptical system between acquiring images of the first surface and theaxially-displaced second surface. In some embodiments, the methodfurther comprises imaging two or more fields-of-view on at least one ofthe first surface or axially-displaced second surface. In someembodiments, the first surface and the axially-displaced second surfacecomprise two surfaces of a flow cell. In some embodiments, said twosurfaces of the flow cell are coated with a hydrophilic coating layer.In some embodiments, said hydrophilic coating layer further compriseslabeled nucleic acid colonies disposed thereon at a surface densityof >10,000 nucleic acid colonies/mm². In some embodiments, an image of asurface of said two surfaces acquired using said optical system shows acontrast to noise ratio (CNR) of at least 5 when the nucleic acidcolonies are labeled with cyanine dye 3 (Cy3), the optical systemcomprises a dichroic mirror and bandpass filter set optimized for Cy3emission, and the image is acquired under non-signal saturatingconditions while the surface is immersed in 25 mM ACES, pH 7.4 buffer.In some embodiments, said optical system comprises 1, 2, 3, or 4 imagingchannels configured to detect nucleic acid colonies disposed on at leastone of the first surface and the axially-displaced second surface thathave been labeled with 1, 2, 3, or 4 distinct detectable labels. In someembodiments, the at least one image sensor comprises pixels having apixel dimension chosen such that a spatial sampling frequency for theoptical system is at least twice an optical resolution of the opticalsystem. In some embodiments, the optical system comprises at least onetube lens positioned between the objective lens and the at least oneimage sensor, and wherein the at least one tube lens is configured tocorrect an imaging performance metric for imaging a first interiorsurface of a flow cell and a second interior surface of the flow cell.In some embodiments, the flow cell has a wall thickness of at least 700um and a gap between the first interior surface and the second interiorsurface of at least 50 um. In some embodiments, the at least one tubelens comprises, in order, an asymmetric convex-convex lens, aconvex-plano lens, an asymmetric concave-concave lens, and an asymmetricconvex-concave lens. In some embodiments, the optical system comprisestwo or more tube lenses which are designed to provide optimal imagingperformance at two or more fluorescence wavelengths. In someembodiments, a combination of objective lens and tube lens is configuredto optimize a modulation transfer function in the mid to high spatialfrequency range. In some embodiments, the imaging performance metriccomprises a measurement of modulation transfer function (MTF) at one ormore specified spatial frequencies, defocus, spherical aberration,chromatic aberration, coma, astigmatism, field curvature, imagedistortion, image contrast-to-noise ratio (CNR), or any combinationthereof. In some embodiments, the optical resolution of images of thefirst surface and axially-displaced second surface arediffraction-limited across the entire field-of-view (FOV). In someembodiments, the sequencing of the nucleic acid molecule furthercomprises performing a sequencing-by-avidity, sequencing-by-nucleotidebase-pairing, sequencing-by-nucleotide binding, orsequencing-by-nucleotide incorporation reaction on at least one of thefirst surface and axially-displaced second surface and detecting a boundor incorporated nucleotide base. In some embodiments, the method furthercomprises determining a genotype of a sample, wherein determining thegenotype of the sample comprises preparing said nucleic acid moleculefor sequencing, and then sequencing said nucleic acid molecule.

Disclosed herein are imaging systems configured to image two distinct,axially-displaced surfaces, the imaging systems comprising an objectivelens and at least one image sensor, wherein said imaging system has anumerical aperture (NA) of less than 0.6 and a field-of-view (FOV) ofgreater than 1.0 mm2, and wherein said imaging system is capable ofacquiring images of the two distinct, axially-displaced surfaces thathave substantially the same optical resolution without moving an opticalcompensator into an optical path between said objective lens and said atleast one image sensor.

In some embodiments, the imaging system has a numerical aperture ofgreater than 0.3. In some embodiments, the imaging system furthercomprises a focusing mechanism used to refocus the optical systembetween acquiring images of the two distinct, axially-displacedsurfaces. In some embodiments, the imaging system is configured to imagetwo or more fields-of-view on at least one of said two distinct,axially-displaced surfaces. In some embodiments, said two distinct,axially-displaced surfaces comprise two surfaces of a flow cell. In someembodiments, said two distinct surfaces of the flow cell are coated witha hydrophilic coating layer, and wherein said hydrophilic coating layerfurther comprises labeled nucleic acid colonies disposed thereon at asurface density of >10,000 nucleic acid colonies/mm². In someembodiments, said imaging system comprises 1, 2, 3, or 4 imagingchannels configured to detect nucleic acid colonies disposed on at leastone of said two distinct surfaces that have been labeled with 1, 2, 3,or 4 distinct detectable labels. In some embodiments, the at least oneimage sensor comprises pixels having a pixel dimension chosen such thata spatial sampling frequency for the imaging system is at least twice anoptical resolution of the imaging system. In some embodiments, theimaging system comprises at least one tube lens positioned between theobjective lens and the at least one image sensor, and wherein the atleast one tube lens is configured to correct an imaging performancemetric for imaging a first interior surface of a flow cell and a secondinterior surface of the flow cell. In some embodiments, the flow cellhas a wall thickness of at least 700 μm and a gap between the firstinterior surface and the second interior surface of at least 50 μm. Insome embodiments, the imaging system comprises two or more tube lenseswhich are designed to provide optimal imaging performance at two or morefluorescence wavelengths. In some embodiments, the optical resolution ofimages of the two distinct, axially-displaced surfaces arediffraction-limited across the entire field-of-view (FOV).

Disclosed herein are methods of sequencing a nucleic acid molecule, themethod comprising: a) imaging a first surface and an axially-displacedsecond surface using a compensation-free optical system which comprisesan objective lens and at least one image sensor, wherein said opticalsystem has a numerical aperture (NA) of less than 0.6 and afield-of-view (FOV) of greater than 1.0 mm²; b) processing the images ofthe first surface and the axially-displaced second surface to correctfor optical aberration such that the images of the first surface and theaxially-displaced second surface have substantially the same opticalresolution; and c) detecting a fluorescently-labeled compositioncomprising the nucleic acid molecule, or a complement thereof, disposedon the first surface or the axially-displaced second surface todetermine an identity of a nucleotide in the nucleic acid molecule.

In some embodiments, the images of the first surface and theaxially-displaced second surface are acquired without moving an opticalcompensator into an optical path between said objective lens and said atleast one image sensor. In some embodiments, the images of the firstsurface and the axially-displaced second surface are acquired by justrefocusing the optical system. In some embodiments, the method furthercomprises imaging two or more fields-of-view on at least one of thefirst surface or axially-displaced second surface. In some embodiments,the first surface and the axially-displaced second surface comprise twosurfaces of a flow cell. In some embodiments, said two surfaces of theflow cell are coated with a hydrophilic coating layer. In someembodiments, said hydrophilic coating layer further comprises labelednucleic acid colonies disposed thereon at a surface density of >10,000nucleic acid colonies/mm². In some embodiments, an image of a surface ofsaid two surfaces acquired using said optical system shows a contrast tonoise ratio (CNR) of at least 5 when the nucleic acid colonies arelabeled with cyanine dye 3 (Cy3), the optical system comprises adichroic mirror and bandpass filter set optimized for Cy3 emission, andthe image is acquired under non-signal saturating conditions while thesurface is immersed in 25 mM ACES, pH 7.4 buffer. In some embodiments,said optical system comprises 1, 2, 3, or 4 imaging channels configuredto detect nucleic acid colonies disposed on at least one of the firstsurface and the axially-displaced second surface that have been labeledwith 1, 2, 3, or 4 distinct detectable labels. In some embodiments, atleast one image sensor comprises pixels having a pixel dimension chosensuch that a spatial sampling frequency for the optical system is atleast twice an optical resolution of the optical system. In someembodiments, the optical system comprises at least one tube lenspositioned between the objective lens and the at least one image sensor,and wherein the at least one tube lens is configured to correct animaging performance metric for imaging a first interior surface of aflow cell and a second interior surface of the flow cell. In someembodiments, the flow cell has a wall thickness of at least 700 μm and agap between the first interior surface and the second interior surfaceof at least 50 μm. In some embodiments, the at least one tube lenscomprises, in order, an asymmetric convex-convex lens, a convex-planolens, an asymmetric concave-concave lens, and an asymmetricconvex-concave lens. In some embodiments, the optical system comprisestwo or more tube lenses which are designed to provide optimal imagingperformance at two or more fluorescence wavelengths. In someembodiments, a combination of objective lens and tube lens is configuredto optimize a modulation transfer function in the mid to high spatialfrequency range. In some embodiments, the imaging performance metriccomprises a measurement of modulation transfer function (MTF) at one ormore specified spatial frequencies, defocus, spherical aberration,chromatic aberration, coma, astigmatism, field curvature, imagedistortion, image contrast-to-noise ratio (CNR), or any combinationthereof. In some embodiments, the optical resolution of images of thefirst surface and axially-displaced second surface arediffraction-limited across the entire field-of-view (FOV). In someembodiments, the sequencing of the nucleic acid molecule furthercomprises performing a sequencing-by-avidity, sequencing-by-nucleotidebinding, or sequencing-by-nucleotide incorporation reaction on at leastone of the first surface and axially-displaced second surface anddetecting a bound or incorporated nucleotide base. In some embodiments,the method further comprises determining a genotype of a sample, whereindetermining the genotype of the sample comprises preparing said nucleicacid molecule for sequencing, and then sequencing said nucleic acidmolecule.

Disclosed herein are systems for sequencing a nucleic acid moleculecomprising: a) an optical system comprising an objective lens and atleast one image sensor, wherein said optical system has a numericalaperture (NA) of less than 0.6 and a field-of-view (FOV) of greater than1.0 mm², and is configured to acquire images of a first surface and anaxially-displaced second surface; and b) a processor programmed to: i)process images of the first surface and the axially-displaced secondsurface to correct for optical aberration such that the images of thefirst surface and the axially-displaced second surface havesubstantially the same optical resolution; and ii) detect afluorescently-labeled composition comprising the nucleic acid molecule,or a complement thereof, disposed on the first surface or theaxially-displaced second surface to determine an identity of anucleotide in the nucleic acid molecule.

In some embodiments, the images of the first surface and theaxially-displaced second surface are acquired without moving an opticalcompensator into an optical path between said objective lens and said atleast one image sensor. In some embodiments, the images of the firstsurface and the axially-displaced second surface are acquired by justrefocusing the optical system. In some embodiments, the imaging systemhas a numerical aperture of greater than 0.3. In some embodiments, thefirst surface and axially-displaced second surface comprise two surfacesof a flow cell. In some embodiments, said two surfaces of the flow cellare coated with a hydrophilic coating layer, and wherein saidhydrophilic coating layer further comprises labeled nucleic acidcolonies disposed thereon at a surface density of >10,000 nucleic acidcolonies/mm². In some embodiments, said optical system comprises 1, 2,3, or 4 imaging channels configured to detect nucleic acid coloniesdisposed on at least one of the first surface or axially-displacedsecond surface that have been labeled with 1, 2, 3, or 4 distinctdetectable labels. In some embodiments, the at least one image sensorcomprises pixels having a pixel dimension chosen such that a spatialsampling frequency for the optical system is at least twice an opticalresolution of the optical system. In some embodiments, the systemcomprises at least one tube lens positioned between the objective lensand the at least one image sensor, and wherein the at least one tubelens is configured to correct an imaging performance metric for imaginga first interior surface of a flow cell and a second interior surface ofthe flow cell. In some embodiments, the flow cell has a wall thicknessof at least 700 μm and a gap between the first interior surface and thesecond interior surface of at least 50 μm. In some embodiments, theoptical system comprises two or more tube lenses which are designed toprovide optimal imaging performance at two or more fluorescencewavelengths.

Disclosed herein are fluorescence imaging systems comprising: a) atleast one light source configured to provide excitation light within oneor more specified wavelength ranges; b) an objective lens configured tocollect fluorescence arising from within a specified field-of-view of asample plane upon exposure of the sample plane to the excitation light,wherein a numerical aperture of the objective lens is at least 0.3,wherein a working distance of the objective lens is at least 700 μm, andwherein the field-of-view has an area of at least 2 mm²; and c) at leastone image sensor, wherein the fluorescence collected by the objectivelens is imaged onto the image sensor, and wherein a pixel dimension forthe image sensor is chosen such that a spatial sampling frequency forthe fluorescence imaging system is at least twice an optical resolutionof the fluorescence imaging system.

In some embodiments, the numerical aperture is at least 0.75. In someembodiments, the numerical aperture is at least 1.0. In someembodiments, the working distance is at least 850 μm. In someembodiments, the working distance is at least 1,000 μm. In someembodiments, the field-of-view has an area of at least 2.5 mm². In someembodiments, the field-of-view has an area of at least 3 mm². In someembodiments, the spatial sampling frequency is at least 2.5 times theoptical resolution of the fluorescence imaging system. In someembodiments, the spatial sampling frequency is at least 3 times theoptical resolution of the fluorescence imaging system. In someembodiments, the system further comprises an X-Y-Z translation stagesuch that the system is configured to acquire a series of two or morefluorescence images in an automated fashion, wherein each image of theseries is acquired for a different field-of-view. In some embodiments, aposition of the sample plane is simultaneously adjusted in an Xdirection, a Y direction, and a Z direction to match the position of anobjective lens focal plane in between acquiring images for differentfields-of-view. In some embodiments, the time required for thesimultaneous adjustments in the X direction, Y direction, and Zdirection is less than 0.4 seconds. In some embodiments, the systemfurther comprises an autofocus mechanism configured to adjust the focalplane position prior to acquiring an image of a different field-of-viewif an error signal indicates that a difference in the position of thefocal plane and the sample plane in the Z direction is greater than aspecified error threshold. In some embodiments, the specified errorthreshold is 100 nm. In some embodiments, the specified error thresholdis 50 nm. In some embodiments, the system comprises three or more imagesensors, and wherein the system is configured to image fluorescence ineach of three or more wavelength ranges onto a different image sensor.In some embodiments, a difference in the position of a focal plane foreach of the three or more image sensors and the sample plane is lessthan 100 nm. In some embodiments, a difference in the position of afocal plane for each of the three or more image sensors and the sampleplane is less than 50 nm. In some embodiments, the total time requiredto reposition the sample plane, adjust focus if necessary, and acquirean image is less than 0.4 seconds per field-of-view. In someembodiments, the total time required to reposition the sample plane,adjust focus if necessary, and acquire an image is less than 0.3 secondsper field-of-view.

Also discloser herein are fluorescence imaging systems for dual-sideimaging of a flow cell comprising: a) an objective lens configured tocollect fluorescence arising from within a specified field-of-view of asample plane within the flow cell; b) at least one tube lens positionedbetween the objective lens and at least one image sensor, wherein the atleast one tube lens is configured to correct an imaging performancemetric for a combination of the objective lens, the at least one tubelens, and the at least one image sensor when imaging an interior surfaceof the flow cell, and wherein the flow cell has a wall thickness of atleast 700 μm and a gap between an upper interior surface and a lowerinterior surface of at least 50 μm; wherein the imaging performancemetric is substantially the same for imaging the upper interior surfaceor the lower interior surface of the flow cell without moving an opticalcompensator into or out of an optical path between the flow cell and theat least one image sensor, without moving one or more optical elementsof the tube lens along the optical path, and without moving one or moreoptical elements of the tube lens into or out of the optical path.

In some embodiments, the objective lens is a commercially-availablemicroscope objective. In some embodiments, the commercially-availablemicroscope objective has a numerical aperture of at least 0.3. In someembodiments, the objective lens has a working distance of at least 700μm. In some embodiments, the objective lens is corrected to compensatefor a cover slip thickness (or flow cell wall thickness) of 0.17 mm. Insome embodiments, the fluorescence imaging system further comprising anelectro-optical phase plate positioned adjacent to the objective lensand between the objective lens and the tube lens, wherein theelectro-optical phase plate provides correction for optical aberrationscaused by a fluid filling the gap between the upper interior surface andthe lower interior surface of the flow cell. In some embodiments, the atleast one tube lens is a compound lens comprising three or more opticalcomponents. In some embodiments, the at least one tube lens is acompound lens comprising four optical components. In some embodiments,the four optical components comprise, in order, a first asymmetricconvex-convex lens, a second convex-plano lens, a third asymmetricconcave-concave lens, and a fourth asymmetric convex-concave lens. Insome embodiments, the at least one tube lens is configured to correct animaging performance metric for a combination of the objective lens, theat least one tube lens, and the at least one image sensor when imagingan interior surface of a flow cell having a wall thickness of at least 1mm. In some embodiments, the at least one tube lens is configured tocorrect an imaging performance metric for a combination of the objectivelens, the at least one tube lens, and the at least one image sensor whenimaging an interior surface of a flow cell having a gap of at least 100μm. In some embodiments, the at least one tube lens is configured tocorrect an imaging performance metric for a combination of the objectivelens, the at least one tube lens, and the at least one image sensor whenimaging an interior surface of a flow cell having a gap of at least 200μm. In some embodiments, the system comprises a single objective lens,two tube lenses, and two image sensors, and each of the two tube lensesis designed to provide optimal imaging performance at a differentfluorescence wavelength. In some embodiments, the system comprises asingle objective lens, three tube lenses, and three image sensors, andeach of the three tube lenses is designed to provide optimal imagingperformance at a different fluorescence wavelength. In some embodiments,the system comprises a single objective lens, four tube lenses, and fourimage sensors, and each of the four tube lenses is designed to provideoptimal imaging performance at a different fluorescence wavelength. Insome embodiments, the design of the objective lens or the at least onetube lens is configured to optimize the modulation transfer function inthe mid to high spatial frequency range. In some embodiments, theimaging performance metric comprises a measurement of modulationtransfer function (MTF) at one or more specified spatial frequencies,defocus, spherical aberration, chromatic aberration, coma, astigmatism,field curvature, image distortion, contrast-to-noise ratio (CNR), or anycombination thereof. In some embodiments, the difference in the imagingperformance metric for imaging the upper interior surface and the lowerinterior surface of the flow cell is less than 10%. In some embodiments,the difference in imaging performance metric for imaging the upperinterior surface and the lower interior surface of the flow cell is lessthan 5%. In some embodiments, the use of the at least one tube lensprovides for an at least equivalent or better improvement in the imagingperformance metric for dual-side imaging compared to that for aconventional system comprising an objective lens, a motion-actuatedcompensator, and an image sensor. In some embodiments, the use of the atleast one tube lens provides for an at least 10% improvement in theimaging performance metric for dual-side imaging compared to that for aconventional system comprising an objective lens, a motion-actuatedcompensator, and an image sensor.

Disclosed herein are illumination systems for use in imaging-basedsolid-phase genotyping and sequencing applications, the illuminationsystem comprising: a) a light source; and b) a liquid light-guideconfigured to collect light emitted by the light source and deliver itto a specified field-of-illumination on a support surface comprisingtethered biological macromolecules.

In some embodiments, the illumination system further comprises acondenser lens. In some embodiments, the specified field-of-illuminationhas an area of at least 2 mm². In some embodiments, the light deliveredto the specified field-of-illumination is of uniform intensity across aspecified field-of-view for an imaging system used to acquire images ofthe support surface. In some embodiments, the specified field-of-viewhas an area of at least 2 mm². In some embodiments, the light deliveredto the specified field-of-illumination is of uniform intensity acrossthe specified field-of-view when a coefficient of variation (CV) forlight intensity is less than 10%. In some embodiments, the lightdelivered to the specified field-of-illumination is of uniform intensityacross the specified field-of-view when a coefficient of variation (CV)for light intensity is less than 5%. In some embodiments, the lightdelivered to the specified field-of-illumination has a speckle contrastvalue of less than 0.1. In some embodiments, the light delivered to thespecified field-of-illumination has a speckle contrast value of lessthan 0.05.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1B schematically illustrate non-limiting examples of imagingdual surface support structures for presenting sample sites for imagingby the imaging systems disclosed herein. FIG. 1A: illustration ofimaging front and rear interior surfaces of a flow cell. FIG. 1B:illustration of imaging front and rear exterior surfaces of a substrate.

FIGS. 2A-2B illustrate a non-limiting example of a multi-channelfluorescence imaging module comprising a dichroic beam splitter fortransmitting an excitation light beam to a sample, and for receiving andredirecting by reflection the resultant fluorescence emission to fourdetection channels configured for detection of fluorescence emission atfour different respective wavelengths or wavelength bands. FIG. 2A: topisometric view. FIG. 2B: bottom isometric view.

FIGS. 3A-3B illustrate the optical paths within the multi-channelfluorescence imaging module of FIGS. 2A and 2B comprising a dichroicbeam splitter for transmitting an excitation light beam to a sample, andfor receiving and redirecting by reflection a resultant fluorescenceemission to four detection channels for detection of fluorescenceemission at four different respective wavelengths or wavelength bands.FIG. 3A: top view. FIG. 3B: side view.

FIG. 4 is a graph illustrating a relationship between dichroic filterperformance and beam angle of incidence.

FIG. 5 is a graph illustrating a relationship between beam footprintsize and beam angle of incidence on a dichroic filter.

FIGS. 6A-6B schematically illustrate an example configuration ofdichroic filters and detection channels of a multi-channel fluorescenceimaging module wherein the dichroic filters have reflective surfacetilted such that the angle between the incident beam (e.g., the centralangle) and the reflective surface of the dichroic filter is less than45. FIG. 6A: schematic illustration of a multichannel fluorescenceimaging module comprising four detection channels. FIG. 6B: detail viewillustrating the angle of incidence (AOI) of a light beam on a dichroicreflector.

FIG. 7 provides a graph illustrating improved dichroic filterperformance corresponding to the imaging module configurationillustrated in FIGS. 6A and 6B.

FIG. 8 provides a graph illustrating improved dichroic filterperformance corresponding to the imaging module configurationillustrated in FIGS. 6A and 6B.

FIGS. 9A-9B provide graphs illustrating reduced surface deformationresulting from the imaging module configuration of FIGS. 6A and 6B. FIG.9A illustrates the effect of folding angle on image quality degradationinduced by the addition of 1 wave of PV spherical power to the lastmirror. FIG. 9B illustrates the effect of folding angle on image qualitydegradation induced by the addition of 0.1 wave of PV spherical power tothe last mirror.

FIGS. 10A-10B provide graphs illustrating improved excitation filterperformance (e.g. sharper transitions between pass bands and surroundingstop bands) resulting from use of s-polarization of the excitation beam.FIG. 10A: transmission spectra for an example bandpass dichroic filterat angles of incidence of 40 degrees and 45 degrees, where the incidentbeam is linearly polarized and is p-polarized with respect to the planeof the dichroic filter. FIG. 10B: changing the orientation of the lightsource with respect to the dichroic filter, such that the incident beamis s-polarized with respect to the plane of the dichroic filter, resultsin a substantially sharper edge between the passband and the stopband.

FIGS. 11A-11B illustrate the modulation transfer function (MTF) of anexample dual surface imaging system disclosed herein having a numericalaperture (NA) of 0.3. FIG. 11A: first surface. FIG. 11B: second surface.

FIGS. 12A-12B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.4. FIG. 12A: first surface.FIG. 12B: second surface.

FIGS. 13A-13B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.5. FIG. 13A: first surface.FIG. 13B: second surface.

FIGS. 14A-14B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.6. FIG. 14A: first surface.FIG. 14B: second surface.

FIGS. 15A-15B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.7. FIG. 15A: first surface.FIG. 15B: second surface.

FIGS. 16A-16B illustrate the MTF of an example dual surface imagingsystem disclosed herein having an NA of 0.8. FIG. 16A: first surface.FIG. 16B: second surface.

FIGS. 17A-17B provide plots of the calculated Strehl ratio for imaging asecond flow cell surface through a first flow cell surface. FIG. 17A:plot of the Strehl ratios for imaging a second flow cell surface througha first flow cell surface as a function of the thickness of theintervening fluid layer (fluid channel height) for different objectivelens and/or optical system numerical apertures. FIG. 17B: plot of theStrehl ratio as a function of numerical aperture for imaging a secondflow cell surface through a first flow cell surface and an interveninglayer of water having a thickness of 0.1 mm.

FIG. 18 provides a schematic illustration of a dual-wavelengthexcitation/four channel emission fluorescence imaging system of thepresent disclosure.

FIG. 19 provides an optical ray tracing diagram for an objective lensdesign that has been designed for imaging a surface on the opposite sideof a 0.17 mm thick coverslip.

FIG. 20 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.17 mm thickcoverslip.

FIG. 21 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.3 mm thickcoverslip.

FIG. 22 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid.

FIG. 23 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface on the opposite side of a 1.0 mm thickcoverslip.

FIG. 24 provides a plot of the modulation transfer function for theobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid.

FIG. 25 provides a ray tracing diagram for a tube lens design which, ifused in conjunction with the objective lens illustrated in FIG. 19,provides for improved dual-side imaging through a 1 mm thick coverslip.

FIG. 26 provides a plot of the modulation transfer function for thecombination of objective lens and tube lens illustrated in FIG. 25 as afunction of spatial frequency when used to image a surface on theopposite side of a 1.0 mm thick coverslip.

FIG. 27 provides a plot of the modulation transfer function for thecombination of objective lens and tube lens illustrated in FIG. 25 as afunction of spatial frequency when used to image a surface that isseparated from that on the opposite side of a 1.0 mm thick coverslip bya 0.1 mm thick layer of aqueous fluid.

FIG. 28 provides ray tracing diagrams for tube lens design (left) of thepresent disclosure that has been optimized to provide high-quality,dual-side imaging performance. Because the tube lens is no longerinfinity-corrected, an appropriately designed null lens (right) may beused in combination with the tube lens to compensate for thenon-infinity-corrected tube lens for manufacturing and testing purposes.

FIG. 29 illustrates one non-limiting example of a single capillary flowcell having 2 fluidic adaptors.

FIG. 30 illustrates one non-limiting example of a flow cell cartridgecomprising a chassis, fluidic adapters, and optionally other components,that is designed to hold two capillaries.

FIG. 31 illustrates one non-limiting example of a system comprising asingle capillary flow cell connected to various fluid flow controlcomponents, where the single capillary is compatible with mounting on amicroscope stage or in a custom imaging instrument for use in variousimaging applications.

FIG. 32 illustrates one non-limiting example of a system that comprisesa capillary flow cell cartridge having integrated diaphragm valves toreduce or minimize dead volume and conserve certain key reagents.

FIG. 33 illustrates one non-limiting example of a system that comprisesa capillary flow cell, a microscope setup, and a temperature controlmechanism.

FIG. 34 illustrates one non-limiting example for temperature control ofthe capillary flow cells through the use of a metal plate that is placedin contact with the flow cell cartridge.

FIG. 35 illustrates one non-limiting approach for temperature control ofthe capillary flow cells that comprises a non-contact thermal controlmechanism.

FIGS. 36A-36C illustrates non-limiting examples of flow cell devicefabrication. FIG. 36A shows the preparation of one-piece glass flowcell. FIG. 36B shows the preparation of two-piece glass flow cell. FIG.36C shows the preparation of three-piece glass flow cell.

FIGS. 37A-37C illustrates non-limiting examples of glass flow celldesigns. FIG. 37A shows a one-piece glass flow cell design. FIG. 37Bshows a two-piece glass flow cell design. FIG. 37C shows a three-pieceglass flow cell design.

FIG. 38 illustrates visualization of cluster amplification in acapillary lumen.

FIG. 39 provides a non-limiting example of a block diagram for asequencing system as disclosed herein.

FIG. 40 provides a non-limiting example of a flow chart for a sequencingmethod as disclosed herein.

FIG. 41 provides a non-limiting example of a schematic for a structuredillumination system as disclosed herein.

FIG. 42 provides a non-limiting example of a flow chart for acquiringand processing structured illumination images of a flow cell surface asdisclosed herein.

FIGS. 43A-43B provide non-limiting schematic illustrations of amultiplexed read-head as disclosed herein. FIG. 43A: side view of amultiplexed read-head in which individual microfluorimeters areconfigured to image a common surface, e.g., the interior surface of aflow cell. FIG. 43B: top view of a multiplexed read-head illustratingthe imaging paths acquired by individual microfluorimeters of themultiplexed read-head.

FIGS. 44A-44B provide non-limiting schematic illustrations of amultiplexed read-head as disclosed herein. FIG. 44A: side view of amultiplexed read-head in which a first subset of a plurality ofindividual microfluorimeters is configured to image a first surface,e.g., a first interior surface of a flow cell, and a second subset ofthe plurality of individual microfluorimeters is configured to image asecond surface, e.g., a second interior surface of a flow cell. FIG.44B: top view of the multiplexed read-head of FIG. 44A illustrating theimaging paths acquired by individual microfluorimeters of themultiplexed read-head.

DETAILED DESCRIPTION

There is a need for fluorescence imaging methods and systems thatprovide increased optical resolution and improved image quality forgenomics applications that lead to corresponding improvements in genomictesting accuracy. Disclosed herein are optical system designs forhigh-performance fluorescence imaging methods and systems that mayprovide any one or more of improved optical resolution (including highperformance optical resolution), improved image quality, and higherthroughput for fluorescence imaging-based genomics applications. Thedisclosed optical illumination and imaging system designs may provideany one or more of the following advantages: improved dichroic filterperformance, increased uniformity of dichroic filter frequency response,improved excitation beam filtering, larger fields-of-view, increasedspatial resolution, improved modulation transfer, contrast-to-noiseratio, and image quality, higher spatial sampling frequency, fastertransitions between image capture when repositioning the sample plane tocapture a series of images (e.g., of different fields-of-view), improvedimaging system duty cycle, and higher throughput image acquisition andanalysis.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications comprising the use of thickflow cell walls (e.g., wall (or coverslip) thickness >700 um) and fluidchannels (e.g., fluid channel height or thickness of 50-200 um) may beachieved using novel objective lens designs that correct for opticalaberration introduced by imaging surfaces on the opposite side of thickcoverslips and/or fluid channels from the objective.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications comprising the use of thickflow cell walls (e.g., wall (or coverslip) thickness >700 um) and fluidchannels (e.g., fluid channel height or thickness of 50-200 um) may beachieved even when using commercially-available, off-the-shelfobjectives by using a novel tube lens design that, unlike the tube lensin a conventional microscope that simply forms an image at theintermediate image plane, corrects for the optical aberrations inducedby the thick flow cell walls and/or intervening fluid layer incombination with the objective.

In some instances, improvements in imaging performance, e.g., formultichannel (e.g., two-color or four-color) imaging applications, maybe achieved by using multiple tube lenses, one for each imaging channel,where each tube lens design has been optimized for the specificwavelength range used in that imaging channel.

In some instances, improvements in imaging performance, e.g., fordual-side (flow cell) imaging applications, may be achieved by using anelectro-optical phase plate in combination with an objective lens tocompensate for the optical aberrations induced by the layer of fluidseparating the upper (near) and lower (far) interior surfaces of a flowcell. In some instances, this design approach may also compensate forvibrations introduced by, e.g., a motion-actuated compensator that ismoved in or out of the optical path depending on which surface of theflow cell is being imaged.

Various multichannel fluorescence imaging module designs are disclosedthat may include illumination and imaging optical paths comprisingfolded optical paths (e.g., comprising one or more beam splitters orbeam combiners, such as dichroic beam splitters or combiners) thatdirect an excitation light beam to an objective lens, and directemission light transmitted through the objective lens to a plurality ofdetection channels. Some particularly advantageous features of thefluorescence imaging modules described herein include specification ofdichroic filter incidence angles that result in sharper and/or moreuniform transitions between passband and stopband wavelength regions ofthe dichroic filters. Such filters may be included within the foldedoptics and may comprise dichroic beam splitters or combiners. Furtheradvantageous features of the disclosed imaging optics designs mayinclude the position and orientation of one or more excitation lightsources and one or more detection optical paths with respect to theobjective lens and to a dichroic filter that receives the excitationbeam. The excitation beam may also be linearly-polarized and theorientation of the linear polarization may be such that s-polarizedlight is incident on the dichroic reflective surface of the dichroicfilter. Such features may potentially improve excitation beam filteringand/or reduce wavefront error introduced into the emission light beamdue to surface deformation of dichroic filters. The fluorescence imagingmodules described herein may or may not include any of these featuresand may or may not include any of these advantages.

Also described herein are devices and systems configured to analyzelarge numbers of different nucleic acid sequences by imaging, e.g.,arrays of immobilized nucleic acid molecules or amplified nucleic acidclusters formed on flow cell surfaces. The devices and systems describedherein can also be useful in, e.g., performing sequencing forcomparative genomics, tracking gene expression, performing micro RNAsequence analysis, epigenomics, aptamer and phage display librarycharacterization, and for performing other sequencing applications. Thedevices and systems disclosed herein comprise various combinations ofoptical, mechanical, fluidic, thermal, electrical, and computingdevices/aspects. The advantages conferred by the disclosed flow celldevices, cartridges, and systems include, but are not limited to: (i)reduced device and system manufacturing complexity and cost, (ii)significantly lower consumable costs (e.g., as compared to those forcurrently available nucleic acid sequencing systems), (iii)compatibility with typical flow cell surface functionalization methods,(iv) flexible flow control when combined with microfluidic components,e.g., syringe pumps and diaphragm valves, etc., and (v) flexible systemthroughput.

Disclosed herein are capillary flow-cell devices and capillary flow cellcartridges that are constructed from off-the-shelf, disposable, singlelumen (e.g., single fluid flow channel) or multi-lumen capillaries thatmay also comprise fluidic adaptors, cartridge chassis, one or moreintegrated fluid flow control components, or any combination thereof.Also disclosed herein are capillary flow cell-based systems that maycomprise one or more capillary flow cell devices (or microfluidicchips), one or more capillary flow cell cartridges (or microfluidiccartridges), fluid flow controller modules, temperature control modules,imaging modules, or any combination thereof

The design features of some disclosed capillary flow cell devices,cartridges, and systems include, but are not limited to, (i) unitaryflow channel construction, (ii) sealed, reliable, and repetitiveswitching between reagent flows that can be implemented with a simpleload/unload mechanism such that fluidic interfaces between the systemand capillaries are reliably sealed, thereby facilitating capillaryreplacement and system reuse, and enabling precise control of reactionconditions such as reagent concentration, pH, and temperature, (iii)replaceable single fluid flow channel devices or capillary flow cellcartridges comprising multiple flow channels that can be usedinterchangeably to provide flexible system throughput, and (iv)compatibility with a wide variety of detection methods such asfluorescence imaging.

Although the disclosed capillary flow cell devices and systems,capillary flow cell cartridges, capillary flow cell-based systems,microfluidic devices and cartridges, and microfluidic chip-basedsystems, are described primarily in the context of their use for nucleicacid sequencing applications, various aspects of the disclosed devicesand systems may be applied not only to nucleic acid sequencing but alsoto any other type of chemical analysis, biochemical analysis, nucleicacid analysis, cell analysis, or tissue analysis application. It shallbe understood that different aspects of the disclosed methods, devices,and systems can be appreciated individually, collectively, or incombination with each other. Although discussed herein primarily in thecontext of fluorescence imaging (including, e.g., fluorescencemicroscopy imaging, fluorescence confocal imaging, two-photonfluorescence, and the like), it will be understood by those of skill inthe art that many of the disclosed optical design approaches andfeatures are applicable to other imaging modes, e.g., bright-fieldimaging, dark-field imaging, phase contrast imaging, and the like.

Definitions: Unless otherwise defined, all of the technical terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art in the field to which this disclosure belongs.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated.

As used herein, the term ‘about’ a number refers to that number plus orminus 10% of that number. The term ‘about’ when used in the context of arange refers to that range minus 10% of its lowest value and plus 10% ofits greatest value.

As used herein, the phrases “imaging module”, “imaging unit”, “imagingsystem”, “optical imaging module”, “optical imaging unit”, and “opticalimaging system” are used interchangeably, and may comprise components orsub-systems of a larger system that may also include, e.g., fluidicsmodules, temperature control modules, translation stages, robotic fluiddispensing and/or microplate handling, processor or computers,instrument control software, data analysis and display software, etc.

As used herein, the term “detection channel” refers to an optical path(and/or the optical components therein) within an optical system that isconfigured to deliver an optical signal arising from a sample to adetector. In some instances, a detection channel may be configured forperforming spectroscopic measurements, e.g., monitoring a fluorescencesignal or other optical signal using a detector such as aphotomultiplier. In some instances, a “detection channel” may be an“imaging channel”, i.e., an optical path (and/or the optical componentstherein) within an optical system that is configured to capture anddeliver an image to an image sensor.

As used herein, a “detectable label” may refer to any of a variety ofdetectable labels or tags known to those of skill in the art. Examplesinclude, but are not limited to, chromophores, fluorophores, quantumdots, upconverting phosphors, luminescent or chemiluminescent molecules,radioisotopes, magnetic nanoparticles, mass tags, and the like. In someinstances, a preferred label may comprise a fluorophore.

As used herein, the term “excitation wavelength” refers to thewavelength of light used to excite a fluorescent indicator (e.g., afluorophore or dye molecule) and generate fluorescence. Although theexcitation wavelength is typically specified as a single wavelength,e.g., 620 nm, it will be understood by those of skill in the art thatthis specification refers to a wavelength range or excitation filterbandpass that is centered on the specified wavelength. For example, insome instances, light of the specified excitation wavelength compriseslight of the specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm,±80 nm, or more. In some instances, the excitation wavelength used mayor may not coincide with the absorption peak maximum of the fluorescentindicator.

As used herein, the term “emission wavelength” refers to the wavelengthof light emitted by a fluorescent indicator (e.g., a fluorophore or dyemolecule) upon excitation by light of an appropriate wavelength.Although the emission wavelength is typically specified as a singlewavelength, e.g., 670 nm, it will be understood by those of skill in theart that this specification refers to a wavelength range or emissionfilter bandpass that is centered on the specified wavelength. In someinstances, light of the specified emission wavelength comprises light ofthe specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm,or more. In some instances, the emission wavelength used may or may notcoincide with the emission peak maximum of the fluorescent indicator.

As used herein, fluorescence is ‘specific’ if it arises fromfluorophores that are annealed or otherwise tethered to the surface,such as fluorescently labeled nucleic acid sequences having a region ofreverse complementarity to a corresponding segment of an oligonucleotideadapter on the surface and annealed to said corresponding segment. Thisfluorescence is contrasted with fluorescence arising from fluorophoresnot tethered to the surface through such an annealing process, or insome cases to background florescence of the surface.

As used herein, a “nucleic acid” (also referred to as a “nucleic acidmolecule”, a “polynucleotide”, “oligonucleotide”, ribonucleic acid(RNA), or deoxyribonucleic acid (DNA)) is a linear polymer of two ormore nucleotides joined by covalent internucleosidic link ages, orvariants or functional fragments thereof. In naturally occurringexamples of nucleic acids, the internucleoside linkage is typically aphosphodiester bond. However, other examples optionally comprise otherinternucleoside linkages, such as phosphorothiolate linkages and may ormay not comprise a phosphate group. Nucleic acids include double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA/RNAhybrids, peptide-nucleic acids (PNAs), hybrids between PNAs and DNA orRNA, and may also include other types of nucleic acid modifications.

As used herein, a “nucleotide” refers to a nucleotide, nucleoside, oranalog thereof. In some cases, the nucleotide is an N- or C-glycoside ofa purine or pyrimidine base (e.g., a deoxyribonucleoside containing2-deoxy-D-ribose or ribonucleoside containing D-ribose). Examples ofother nucleotide analogs include, but are not limited to,phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methylphosphonates, 2-0-methyl ribonucleotides, and the like.

Fluorescence imaging viewed as an information pipeline: A usefulabstraction of the role that fluorescence imaging systems plays intypical genomic assay techniques (including nucleic acid sequencingapplications) is as an information pipeline, where the photon signalenters at one end of the pipeline, e.g., the objective lens used forimaging, and location specific information regarding the fluorescencesignal emerges at the other end of the pipeline, e.g., at the positionof the image sensor. When more information is pumped through thispipeline, some content, inevitably, will be lost during this transferprocess and never recovered. An example of this case is when too manylabeled molecules (or clonally-amplified clusters of molecules) arepresent within a small region of a substrate surface to be clearlyresolved in the image; at the position of the image sensor, it becomesdifficult to differentiate photon signals arising from adjacent clustersof molecules, thus increasing the probability of attributing the signalto the wrong cluster and leading to detection errors.

Design of optical imaging modules: The goal of designing an opticalimaging module is thus to maximize the flow of information contentthrough this detection pipeline and to minimize detection errors.Several key design elements need to be addressed in the design process,including:

1) Matching the physical feature density on the substrate surface to beimaged with the overall image quality of the optical imaging system andthe pixel sampling frequency of the image sensor used. A mismatch ofthese parameters may result in loss of information or sometimes even thegeneration of false information, e.g., spatial aliasing may arise whenpixel sampling frequency is lower than twice the optical resolutionlimit.

2) Matching the size of the area to be imaged with the overall imagequality of the optical imaging system and focus quality across theentire field of view.

3) Matching the optical collection efficiency, modulation transferfunction, and image sensor performance characteristics of the opticalsystem design with the fluorescence photon flux expected for the inputexcitation photon flux, dye efficiency (related to dye extinctioncoefficient and fluorescence quantum yield), while accounting forbackground signal and system noise characteristics.

4) Maximizing the separation of spectral content to reduce cross talkbetween fluorescence imaging channels.

5) Effective synchronization of image acquisition steps withrepositioning of the sample or optics between image capture of differentfields-of-view to minimize the down time (or maximize the duty cycle) ofthe imaging system and thus maximize the overall throughput of the imagecapture process.

This disclosure describes a systematic way to address each of the designelements outlined above and to create component level specifications forthe imaging system.

Improved optical resolution and image quality to improve or maximizeinformation transfer and throughput: One non-limiting design practicemay be to start with the optical resolution required to distinguish twoadjacent features as specified in terms of a number, X, of line pairsper mm (lp/mm) and translate it to a corresponding numerical aperture(NA) requirement. The numerical aperture requirement can then be used toassess the resulting impact on modulation transfer function and imagecontrast.

The standard modulation transfer function (MTF) describes the spatialfrequency response for image contrast (modulation) transferred throughan optical system; image contrast decreases as a function of spatialfrequency and increases with increasing NA. This function limits thecontrast/modulation that can be achieved for a given NA. Furthermore,wave front error can negatively impact the MTF, thus making it desirableto improve or optimize the optical system design using the true systemMTF instead of that predicted by diffraction-limited optics. Note that,as used herein, MTF will refer to the total system MTF (including thecomplete optical path from coverslip to image sensor) although designpractice may primarily consider the MTF of the objective lens.

In genomic testing applications, where the target to be imaged is anarray of high density “spots” on a surface (either randomly distributedor patterned), one can determine the minimum modulation transfer valuerequired by downstream analysis to resolve two adjacent spots anddiscriminate between four possible states (e.g., ON-OFF, ON-ON, OFF-ONand OFF-OFF). For example, assume that the spots are small enough to beapproximated as point sources of light. Assuming that the detection taskis to determine if the two adjacent spots separated by a distance, d,are ON or OFF (in other words, bright or dark), and that thecontrast-to-noise ratio (CNR) for the fluorescence signals arising fromthe spots at the sample plane (or object plane) is Csampie, then underideal conditions the CNR of the readout signal for the two adjacentspots at the image sensor plane, C_(image), can be closely approximatedas C_(image)=C_(sample)* MTF(1/d), where MTF(1/d) is the MTF value atspatial frequency=(1/d).

In a typical design, the value of C may need to be at least 4 so that asimple threshold method can be used to avoid misclassification offluorescence signals. Assuming a Gaussian distribution of fluorescencesignal intensities around a mean value, at C_(image)=4, the expectederror in correctly classifying fluorescence signals (e.g., as being ONor OFF) is <0.035%. The use of proprietary high CNR sequencing andsurface chemistry, such as that described in U.S. patent applicationSer. No. 16/363,842, allows one to achieve sample plane CNR (C_(sample))values for clusters of clonally-amplified, labeled oligonucleotidemolecules tethered to a substrate surface of greater than 12 (or evenmuch higher) when measured for a sparse field (i.e., at a low surfacedensity of clusters or spots) where the MTF has a value of close to100%. Assuming a sample plane CNR value of C_(sample)>12 and targeting aclassification error rate of <0.1% (thus, C_(image)>4), in someimplementations the minimum value for M(1/d) can be determined as M(1/d)=4/12˜33%. Thus, a modulation transfer function threshold of at least33% may be used to retain the information content of the transferredimage.

Design practice can relate the minimum separation distance of twofeatures or spots, d, to the optical resolution requirement (specifiedas noted above in terms of X (lp/mm)) as d =(1 mm)/X, i.e., d is theminimum separation distance between two features or spots which can befully resolved by the optical system. In some designs disclosed herein,where the objective of the design analysis is to increase or maximizerelevant information transfer, this design criterion can be relaxed tod=(1 mm)/X/A, where 2>A>1. For the same optical resolution of X 1 p/mm,the value of d, the minimum resolvable spot separation distance at thesample plane, is reduced, thereby enabling the use of higher featuredensities.

Design practice determines the minimum spatial sampling frequency at thesample plane using the Nyquist criteria, where spatial samplingfrequency S>2*X (and where X is the optical resolution of the imagingsystem specified in terms of X 1p/mm as noted above). When the systemspatial sampling frequency is close to the Nyquist criteria, as is oftenthe case, imaging system resolution of greater than S results inaliasing as the higher frequency information resolved by the opticalsystem cannot be sufficiently sampled by the image sensor.

In the some of the designs disclosed herein, an oversampling schemebased on the relationship S=B*Y (where B≥2 and Y is the true opticalsystem MTF limit) may be used to further improve the informationtransfer capacity of the imaging system. As indicated above, X (1p/mm)corresponds to a practical, non-zero (>33%) minimum modulation transfervalue, whereas Y (1p/mm) is the limit of optical resolution somodulation at Y(1p/mm) is 0. Thus, in the disclosed designs, Y (lp/mm)may advantageously be significantly greater than X For values of B≥2,the disclosed designs are oversampling for the sample object frequencyX, i.e., S≥B*Y>2*X

The above relationship can be used to determine the system magnificationand may provide an upper bound for image sensor pixel size. The choiceof image sensor pixel size is matched to the system optical quality aswell to the spatial sampling frequency required to reduce aliasing. Thelower bound of image sensor pixel size can be determined based on photonthroughput, as relative noise contributions increase with smallerpixels.

Other design approaches are, however, also possible. For example,reducing the NA to less than 0.6 (e.g., 0.5 or less,) may provideincreased depth of field. Such increased depth of field may enable dualsurface imagining wherein two surfaces at different depths can be imagedat the same time with or without refocusing. As discussed above,reducing NA may reduce optical resolution. In some implementations, useof higher excitation beam power, e.g., 1 W or higher, may be employed toproduce strong signal. An inherently high contrast sample (i.e.,comprising a sample surface that exhibits strong foreground signal anddramatically reduced background signal, may also be used to facilitateacquisition of high contrast-to-noise ratio (CNR) images, e.g., havingCNR values of >20, that provide for improved signal discrimination forbase-calling in nucleic acid sequencing applications, etc. In someoptical system designs disclosed herein, sample support structures suchas flow cells having hydrophilic surfaces are used to reduce backgroundnoise.

In various implementations, a large field-of-view (FOV) is provided bythe disclosed optical systems. For example, a FOV of greater than 2 or 3mm may be provided with some optical imaging systems comprising, e.g.,an objective lens and a tube lens. In some cases, the optical imagingsystem provides a reduced magnification, for example, a magnification ofless than 10x. Such reduced magnification may in some implementationsfacilitate large FOV designs. Despite a reduced magnification, theoptical resolution of such systems can still be sufficient as detectorarrays having small pixel size or pitch may be used. In someimplementations, image sensors comprising a pixel size that is smallerthan twice the optical resolution provided by the optical imaging system(e.g., objective and tube lens) may be used to satisfy the Nyquisttheorem.

Still other designs are also possible. In some optical designsconfigured to provide for dual surface imaging where two surfaces atdifferent depths can be imaged at the same time, the optical imagingsystem (e.g., the objective lens and/or tube lens) is configured toreduce optical aberration for imaging said two surfaces (e.g., twoplanes) at those two respective depths more than at other locations(e.g., other planes) at other depths. Additionally, the optical imagingsystem may be configured to reduce aberration for imaging said twosurfaces (e.g., two planes) at those two respective depths through atransmissive layer on said sample support structure (such as a layer ofglass (e.g., a cover slip) and through a solution (e.g., an aqueoussolution) comprising the sample or in contact with a sample on at leastone of said two surfaces.

Multichannel fluorescence imaging modules and systems: In someinstances, the imaging modules or systems disclosed herein may comprisefluorescence imaging modules or systems. In some instances, thefluorescence imaging systems disclosed herein may comprise a singlefluorescence excitation light source (for providing excitation light ata single wavelength or within a single excitation wavelength range) andan optical path configured to deliver the excitation light to a sample(e.g., fluorescently-tagged nucleic acid molecules or clusters thereofdisposed on a substrate surface). In some instances, the fluorescenceimaging systems disclosed herein may comprise a single fluorescenceemission imaging and detection channel, e.g., an optical path configuredto collect fluorescence emitted by the sample and deliver an image ofthe sample (e.g., an image of a substrate surface on whichfluorescently-tagged nucleic acid molecules or clusters thereof aredisposed) to an image sensor or other photodetection device. In someinstances, the fluorescence imaging systems may comprise two, three,four, or more than four fluorescence excitation light sources and/oroptical paths configured to deliver excitation light at two, three,four, or more than four excitation wavelengths (or within two, three,four, or more than four excitation wavelength ranges). In someinstances, the fluorescence imaging systems disclosed herein maycomprise two, three, four, or more than four fluorescence emissionimaging and detection channels configured to collect fluorescenceemitted by the sample at two, three, four, or more than four emissionwavelengths (or within two, three, four, or more than four emissionwavelength ranges and deliver an image of the sample (e.g., an image ofa substrate surface on which fluorescently-tagged nucleic acid moleculesor clusters thereof are disposed) to two, three, four, or more than fourimage sensors or other photodetection devices.

Dual surface imaging: In some instances, the imaging systems disclosedherein, including fluorescence imaging systems, may be configured toacquire high-resolution images of a single sample support structure orsubstrate surface. In some instances, the imaging systems disclosedherein, including fluorescence imaging systems, may be configured toacquire high-resolution images of two or more sample support structuresor substrate surfaces, e.g., two or more surfaces of a flow cell. Insome instances, the high-resolution images provided by the disclosedimaging systems may be used to monitor reactions occurring on the two ormore surfaces of the flow cell (e.g., nucleic acid hybridization,amplification, and/or sequencing reactions) as various reagents flowthrough the flow cell or around a flow cell substrate. FIG. 1A and FIG.1B provide schematic illustrations of such dual surface supportstructures. FIG. 1A shows a dual surface support structure such as aflow cell that includes an internal flow channel through which ananalyte or reagent can be flowed. The flow channel may be formed betweenfirst and second, top and bottom, and/or front and back layers such asfirst and second, top and bottom, and/or front and back plates as shown.One or more of the plates may include a glass plate, such as acoverslip, or the like. In some implementations, the layer comprisesborosilicate glass, quartz, or plastic. Interior surfaces of these topand bottom layers provide walls of the flow channel that assist inconfining the flow of analyte or reagent through the flow channel of theflow cell. In some designs, these interior surfaces are planar.Similarly, the top and bottom layers may be planar. In some designs, atleast one additional layer (not shown) is disposed between the top andbottom layers. This additional layer may have one or more pathways cuttherein that assist in defining one or more flow channels andcontrolling the flow of the analyte or reagent within the flow channel.Additional discussion of sample support structures, e.g., flow cells,can be found below.

FIG. 1A schematically illustrates a plurality of fluorescing samplesites on the first and second, top and bottom, and/or front and backinterior surfaces of the flow cell. In some implementations, reactionsmay occur at these at these sites to bind sample such that fluorescenceis emitted from these sites (note that FIG. 1A is schematic and notdrawn to scale; for example, the size and spacing of the fluorescingsample sites may be smaller than shown).

FIG. 1B shows another dual surface support structure having two surfacescontaining fluorescing sample sites to be imaged. The sample supportstructure comprises a substrate having first and second, top and bottom,and/or front and back exterior surfaces. In some designs, these exteriorsurfaces are planar. In various implementations, the analyte or reagentis flowed across these first and second exterior surfaces. FIG. 1Bschematically illustrates a plurality of fluorescing sample sites on thefirst and second, top and bottom, and/or front and back exteriorsurfaces of the sample support structure. In some implementations,reactions may occur at these at these sites to bind sample such thatfluorescence is emitted from these sites (note that FIG. 1B is schematicand not drawn to scale; for example, the size and spacing of thefluorescing sample sites may be smaller than shown).

In some instances, the fluorescence imaging modules and systemsdescribed herein may be configured to image such fluorescing samplesites on first and second surfaces at different distances from theobjective lens. In some designs, only one of the first or secondsurfaces is in focus at a time. Accordingly, in such designs, one of thesurfaces is imaged at a first time, and the other surface is imaged at asecond time. The focus of the fluorescence imaging module may be changedafter imaging one of the surfaces in order to image the other surfacewith comparable optical resolution, as the images of the two surfacesare not simultaneously in focus. In some designs, an opticalcompensation element may be introduced into the optical path between thesample support structure and the image sensor in order to image one ofthe two surfaces. The depth of field in such fluorescence imagingconfigurations may not be sufficiently large to include both the firstand second surfaces. In some implementations of the fluorescence imagingmodules described herein, both the first and second surfaces may beimaged at the same time, i.e., simultaneously. For example, thefluorescence imaging module may have a depth of field that issufficiently large to include both surfaces. In some instances, thisincreased depth of field may be provided by, for example, reducing thenumerical aperture of the objective lens (or microscope objective) aswill be discussed in more detail below.

As shown in FIGS. 1A and 1B, the imaging optics (e.g., an objectivelens) may be positioned at a suitable distance (e.g., a distancecorresponding to the working distance) from the first and secondsurfaces to form in-focus images of the first and second surfaces on animage sensor of a detection channel. As shown in the example of FIGS. 1Aand 1B, the first surface may be between said objective lens and thesecond surface. For example, as illustrated, the objective lens isdisposed above both the first and second surfaces, and the first surfaceis disposed above the second surface. The first and second surfaces, forexample, are at different depths. The first and second surfaces are atdifferent distances from any one or more of the fluorescence imagingmodule, the illumination and imaging module, imaging optics, or theobjective lens. The first and second surfaces are separated from eachother with the first surface spaced apart above the second surface. Inthe example shown, the first and second surfaces are planar surfaces andare separated from each other along a direction normal to said first andsecond planar surfaces. Also, in the example shown, said objective lenshas an optical axis and said first and second surfaces are separatedfrom each other along the direction of said optical axis. Similarly, theseparation between the first and second surfaces may correspond to thelongitudinal distance such as along the optical path of the excitationbeam and/or along an optical axis through the fluorescence imagingmodule and/or the objective lens. Accordingly, these two surfaces may beseparated by a distance from each other in the longitudinal (Z)direction, which may be along the direction of the central axis of theexcitation beam and/or the optical axis of the objective lens and/or thefluorescence imaging module. This separation may correspond, forexample, to a flow channel within a flow cell in some implementations.

In various designs, the objective lens (possibly in combination withanother optical component, e.g., a tube lens) have a depth of fieldand/or depth of focus that is at least as large as the longitudinalseparation (in the Z direction) between the first and second surfaces.The objective lens, alone or in combination with the additional opticalcomponent, may thus simultaneously form in-focus images of both thefirst and the second surface on an image sensor of one or more detectionchannels where these images have comparable optical resolution. In someimplementations, the imaging module may or may not need to be re-focusedto capture images of both the first and second surfaces with comparableoptical resolution. In some implementations, compensation optics neednot be moved into or out of an optical path of the imaging module toform in-focus images of the first and second surfaces. Similarly, insome implementations, one or more optical elements (e.g., lens elements)in the imaging module (e.g., the objective lens and/or a tube lens) neednot be moved, for example, in the longitudinal direction along the firstand/or second optical paths (e.g., along the optical axis of the imagingoptics) to form in-focus images of the first surface in comparison tothe location of said one or more optical element when used to formin-focus images of the second surface. In some implementations, however,the imaging module includes an autofocus system configured to provideboth the first and second surface in focus at the same time. In variousimplementations, the sample is in focus to sufficiently resolve thesample sites, which are closely spaced together in lateral directions(e.g., the X and Y directions). Accordingly, in various implementations,no optical element enters an optical path between the sample supportstructure (e.g., between a translation stage that supports the samplesupport structure) and an image sensor (or photodetector array) in theat least one detection channel in order to form in-focus images offluorescing sample sites on a first surface of the sample supportstructure and on a second surface of said sample support structure.Similarly, in various implementations, no optical compensation is usedto form an in-focus image of fluorescing sample sites on a first surfaceof the sample support structure on the image sensor or photodetectorarray that is not identical to optical compensation used to form anin-focus image of fluorescing sample sites on a second surface of thesample support structure on the image sensor or photodetector array.Additionally, in certain implementations, no optical element in anoptical path between the sample support structure (e.g., between atranslation stage that supports the sample support structure) and animage sensor in the at least one detection channel is adjusteddifferently to form an in-focus image of fluorescing sample sites on afirst surface of the sample support structure than to form an in-focusimage of fluorescing sample sites on a second surface of the samplesupport structure. Similarly, in some various implementations, nooptical element in an optical path between the sample support structure(e.g., between a translation stage that supports the sample supportstructure) and an image sensor in the at least one detection channel ismoved a different amount or a different direction to form an in-focusimage of fluorescing sample sites on the a first surface of the samplesupport structure on the image sensor than to form an in-focus image offluorescing sample sites on a second surface of said sample supportstructure on the image sensor. Any combination of the features ispossible. For example, in some implementations, in-focus images of theupper interior surface and the lower interior surface of the flow cellcan be obtained without moving an optical compensator into or out of anoptical path between the flow cell and the at least one image sensor andwithout moving one or more optical elements of the imaging system (e.g.,the objective and/or tube lens) along the optical path (e.g., opticalaxis) therebetween. For example, in-focus images of the upper interiorsurface and the lower interior surface of the flow cell can be obtainedwithout moving one or more optical elements of the tube lens into or outof the optical path, or without moving one or more optical elements ofthe tube lens along the optical path (e.g., optical axis) therebetween.

Any one or more of the fluorescence imaging module, the illuminationoptical path, the imaging optical path, the objective lens, or the tubelens may be designed to reduce or minimize optical aberration at twolocations such as two planes corresponding to two surfaces on a flowcell or other sample support structure, for example, where fluorescingsample sites are located. Any one or more of the fluorescence imagingmodule, the illumination optical path, the imaging optical path, theobjective lens, or the tube lens may be designed to reduce or minimizeoptical aberration at the selected locations or planes relative to otherlocations or planes, such as first and second surfaces containingfluorescing sample sites on a dual surface flow cell. For example, anyone or more of the fluorescence imaging module, the illumination opticalpath, the imaging optical path, the objective lens, or the tube lens maybe designed to reduce or minimize optical aberration at two depths orplanes located at different distances from the objective lens ascompared to the aberrations associated with other depths or planes atother distances from the objective lens. For example, optical aberrationmay be less for imaging the first and second surfaces than elsewhere ina region ranging from about 1 to about 10 mm from the objective lens.Additionally, any one or more of the fluorescence imaging module, theillumination optical path, the imaging optical path, the objective lens,or the tube lens may, in some instances, be configured to compensate foroptical aberration induced by transmission of emission light through oneor more portions of the sample support structure such as a layer thatincludes one of the surfaces on which sample adheres as well as possiblya solution that is in contact with the sample. This layer (e.g., acoverslip or the wall of a flow cell) may comprise, e.g., glass, quartz,plastic, or other transparent material having a refractive index andthat introduces optical aberration.

Accordingly, the imaging performance may be substantially the same whenimaging the first surface and second surface. For example, the opticaltransfer functions (OTF) and/or modulation transfer functions (MTF) maybe the substantially the same for imaging of the first and secondsurfaces. Either or both of these transfer functions may, for example,be within 20%, within 15%, within 10%, within 5%, within 2.5%, or within1% of each other, or within any range formed by any of these values atone or more specified spatial frequencies or when averaged over a rangeof spatial frequencies. Accordingly, an imaging performance metric maybe substantially the same for imaging the upper interior surface or thelower interior surface of the flow cell without moving an opticalcompensator into or out of an optical path between the flow cell and theat least one image sensor, and without moving one or more opticalelements of the imaging system (e.g., the objective and/or tube lens)along the optical path (e.g., optical axis) therebetween. For example,an imaging performance metric may be substantially the same for imagingthe upper interior surface or the lower interior surface of the flowcell without moving one or more optical elements of the tube lens intoor out of the optical path or without moving one or more opticalelements of the tube lens along the optical path (e.g., optical axis)therebetween. Additional discussion of MTF is included below and in U.S.Provisional Application No. 62/962,723 filed Jan. 17, 2020, which isincorporated herein by reference in its entirety.

It will be understood by those of skill in the art that the disclosedimaging modules or systems may, in some instances, be stand-aloneoptical systems designed for imaging a sample or substrate surface. Insome instances, they may comprise one or more processors or computers.In some instances, they may comprise one or more software packages thatprovide instrument control functionality and/or image processingfunctionality. In some instances, in addition to optical components suchas light sources (e.g., solid-state lasers, dye lasers, diode lasers,arc lamps, tungsten-halogen lamps, etc.), lenses, prisms, mirrors,dichroic reflectors, beam splitters, optical filters, optical bandpassfilters, light guides, optical fibers, apertures, and image sensors(e.g., complementary metal oxide semiconductor (CMOS) image sensors andcameras, charge-coupled device (CCD) image sensors and cameras, etc.),they may also include mechanical and/or optomechanical components, suchas X-Y translation stages, X-Y-Z translation stages, piezoelecticfocusing mechanisms, electro-optical phase plates, and the like. In someinstances, they may function as modules, components, sub-assemblies, orsub-systems of larger systems designed for, e.g., genomics applications(e.g., genetic testing and/or nucleic acid sequencing applications). Forexample, in some instances, they may function as modules, components,sub-assemblies, or sub-systems of larger systems that further compriselight-tight and/or other environmental control housings, temperaturecontrol modules, flow cells and cartridges, fluidics control modules,fluid dispensing robotics, cartridge- and/or microplate-handling(pick-and-place) robotics, one or more processors or computers, one ormore local and/or cloud-based software packages (e.g., instrument/systemcontrol software packages, image processing software packages, dataanalysis software packages), data storage modules, data communicationmodules (e.g., Bluetooth, WiFi, intranet, or intern& communicationhardware and associated software), display modules, etc., or anycombination thereof. These additional components of larger systems,e.g., systems designed for genomics applications, will be discussed inmore detail below.

FIGS. 2A and 2B illustrate a non-limiting example of an illumination andimaging module 100 for multi-channel fluorescence imaging. Theillumination and imaging module 100 includes an objective lens 110, anillumination source 115, a plurality of detection channels 120, and afirst dichroic filter 130, which may comprise a dichroic reflector orbeam splitter. An autofocus system, which may include an autofocus laser102, for example, that projects a spot the size of which is monitored todetermine when the imaging system is in-focus may be included in somedesigns. Some or all components of the illumination and imaging module100 may be coupled to a baseplate 105.

The illumination or light source 115 may include any suitable lightsource configured to produce light of at least a desired excitationwavelength (discussed in more detail below). The light source may be abroadband source that emits light within one or more excitationwavelength ranges (or bands). The light source may be a narrowbandsource that emits light within one or more narrower wavelength ranges.In some instances, the light source may produce a single isolatedwavelength (or line) corresponding to the desired excitation wavelength,or multiple isolated wavelengths (or lines). In some instances, thelines may have some very narrow bandwidth. Example light sources thatmay be suitable for use in the illumination source 115 include, but arenot limited to, an incandescent filament, xenon arc lamp, mercury-vaporlamp, a light-emitting diode, a laser source such as a laser diode or asolid-state laser, or other types of light sources. As discussed below,in some designs, the light source may comprise a polarized light sourcesuch as a linearly polarized light source. In some implementations, theorientation of the light source is such that s-polarized light isincident on one or more surfaces of one or more optical components suchas the dichroic reflective surface of one or more dichroic filters.

The illumination source 115 may further include one or more additionaloptical components such as lenses, filters, optical fibers, or any othersuitable transmissive or reflective optics as appropriate to output anexcitation light beam having suitable characteristics toward a firstdichroic filter 130. For example, beam shaping optics may be included,for example, to receive light from a light emitter in the light sourceand produce a beam and/or provide a desired beam characteristic. Suchoptics may, for example, comprise a collimating lens configured toreduce the divergence of light and/or increase collimation and/or tocollimate the light.

In some implementations, multiple light sources are included in theillumination and imaging module 100. In some such implementations,different light sources may produce light having different spectralcharacteristics, for example, to excite different fluorescence dyes. Insome implementations, light produced by the different light sources maydirected to coincide and form an aggregate excitation light beam. Thiscomposite excitation light beam may be composed of excitation lightbeams from each of the light sources. The composite excitation lightbeam will have more optical power than the individual beams that overlapto form the composite beam. For example, in some implementations thatinclude two light sources that produce two excitation light beams, thecomposite excitation light beam formed from the two individualexcitation light beams may have optical power that is the sum of theoptical power of the individual beams. Similarly, in someimplementations, three, four, five or more light sources may beincluded, and these light sources may each output excitation light beamsthat together form a composite beam that that has an optical power thatis the sum of the optical power of the individual beams.

In some implementations, the light source 115 outputs a sufficientlylarge amount of light to produce sufficiently strong fluorescenceemission. Stronger fluorescence emission can increase thesignal-to-noise ratio (SNR) and the contrast-to-noise ratio (CNR) ofimages acquired by the fluorescence imaging module. In someimplementations, the output of the light source and/or an excitationlight beam derived therefrom (including a composite excitation lightbeam) may range in power from about 0.5 W to about 5.0 W, or more (aswill be discussed in more detail below).

Referring again to FIGS. 2A and 2B, the first dichroic filter 130 isdisposed with respect to the light source to receive light therefrom.The first dichroic filter may comprise a dichroic mirror, dichroicreflector, dichroic beam splitter, or dichroic beam combiner configuredto transmit light in a first spectral region (or wavelength range) andreflect light having a second spectral region (or wavelength range). Thefirst spectral region may include one or more spectral bands, e.g., oneor more spectral bands in the ultraviolet and blue wavelength ranges.Similarly, a second spectral region may include one or more spectralbands, e.g., one or more spectral bands extending from the green to redand infrared wavelengths. Other spectral regions or wavelength rangesare also possible.

In some implementations, the first dichroic filter may be configured totransmit light from the light source to a sample support structure suchas to a microscope slide, a capillary, a flow cell, a microfluidic chip,or other substrate or support structure. The sample support structuresupports and positions the sample, e.g., a composition comprising afluorescently-labeled nucleic acid molecule or complement thereof, withrespect to the illumination and imaging module 100. Accordingly, a firstoptical path extends from the light source to the sample via the firstdichroic filter. In various implementations, the sample supportstructure includes at least one surface on which the sample is disposedor to which the sample binds. In some instances, the sample may bedisposed within or bound to different localized regions or sites on theat least one surface of the sample support structure.

In some instances, the support structure may include two surfaceslocated at different distances from objective lens 110 (i.e., atdifferent positions or depths along the optical axis of objective lens110) on which the sample is disposed. As discussed below, for example, aflow cell may comprise a fluid channel formed at least in part by firstand second (e.g., upper and lower) interior surfaces, and the sample maybe disposed at localized sites on the first interior surface, the secondinterior surface, or both interior surfaces. The first and secondsurface may be separated by the region corresponding to the fluidchannel through which a solution flows, and thus be at differentdistances or depth with respect to objective lens 110 of theillumination and imaging module 100.

The objective lens 110 may be included in the first optical path betweenthe first dichroic filter and the sample. This objective lens may beconfigured, for example, to have a focal length, working distance,and/or be positioned to focus light from the light source(s) onto thesample, e.g., onto a surface of the microscope slide, capillary, flowcell, microfluidic chip, or other substrate or support structure.Similarly, the objective lens 110 may be configured to have suitablefocal length, working distance, and/or be positioned to collect lightreflected, scattered, or emitted from the sample (e.g., fluorescenceemission) and to form an image of the sample (e.g., a fluorescenceimage).

In some implementations, objective lens 110 may comprise a microscopeobjective such as an off-the-shelf objective. In some implementations,objective lens 110 may comprise a custom objective. An example of acustom objective lens and/or custom objective—tube lens combination isdescribed below and in U.S. Provisional Application No. 62/962,723 filedon Jan. 17, 2020, which is incorporated herein by reference in itsentirety. The objective lens 110 may be designed to reduce or minimizeoptical aberration at two locations such as two planes corresponding totwo surfaces of a flow cell or other sample support structure. Theobjective lens 110 may be designed to reduce the optical aberration atthe selected locations or planes, e.g., the first and second surfaces ofa dual surface flow cell, relative to other locations or planes in theoptical path. For example, the objective lens 110 may be designed toreduce the optical aberration at two depths or planes located atdifferent distances from the objective lens as compared to the opticalaberrations associated with other depths or planes at other distancesfrom the objective. For example, in some instances, optical aberrationmay be less for imaging the first and second surfaces of a flow cellthan that exhibited elsewhere in a region spanning from 1 to 10 mm fromthe front surface of the objective lens. Additionally, a customobjective lens 110 may in some instances be configured to compensate foroptical aberration induced by transmission of fluorescence emissionlight through one or more portions of the sample support structure, suchas a layer that includes one or more of the flow cell surfaces on whicha sample is disposed, or a layer comprising a solution filling the fluidchannel of a flow cell. These layers may comprise, e.g., glass, quartz,plastic, or other transparent material having a refractive index, andwhich may introduce optical aberration.

In some implementations, objective lens 110 may have a numericalaperture (NA) of 0.6 or more (as discussed in more detail below). Such anumerical aperture may provide for reduced depth of focus and/or depthof field, improved background discrimination, and increased imagingresolution.

In some implementations, objective lens 110 may have a numericalaperture (NA) of 0.6 or less (as discussed in more detail below). Such anumerical aperture may provide for increased depth of focus and/or depthof field. Such increased depth of focus and/or depth of field mayincrease the ability to image planes separated by a distance such asthat that separates the first and second surfaces of a dual surface flowcell.

As discussed above, a flow cell may comprise, for example, first andsecond layers comprising first and second interior surfaces respectivelythat are separated by a fluid channel through which an analyte orreagent can flow. In some implementations, the objective lens 110 and/orillumination and imaging module 100 may be configured to provide a depthof field and/or depth of focus sufficiently large to image both thefirst and second interior surfaces of the flow cell, either sequentiallyby re-focusing the imaging module between imaging the first and secondsurfaces, or simultaneously by ensuring a sufficiently large depth offield and/or depth of focus, with comparable optical resolution. In someinstances, the depth of field and/or depth of focus may be at least aslarge or larger than the distance separating the first and secondsurfaces of the flow cell to be imaged, such as the first and secondinterior surfaces of the flow cell. In some instances, the first andsecond surfaces, e.g., the first and second interior surfaces of a dualsurface flow cell or other sample support structure, may be separated,for example, by a distance ranging from about 10 μm to about 700 μm, ormore (as will be discussed in more detail below). In some instances, thedepth of field and/or depth of focus may thus range from about 10 μm toabout 700 μm, or more (as will be discussed in more detail below).

In some designs, compensation optics (e.g., an “optical compensator” or“compensator”) may be moved into or out of an optical path in theimaging module, for example, an optical path by which light collected bythe objective lens 110 is delivered to an image sensor, to enable theimaging module to image the first and second surfaces of the dualsurface flow cell. The imaging module may be configured, for example, toimage the first surface when the compensation optics is included in theoptical path between the objective lens and an image sensor orphotodetector array configured to capture an image of the first surface.In such a design, the imaging module may be configured to image thesecond surface when the compensation optics is removed from or notincluded in the optical path between the objective lens 110 and theimage sensor or photodetector array configured to capture an image ofthe second surface. The need for an optical compensator may be morepronounced when using an objective lens 110 with a high numericalaperture (NA) value, e.g., for numerical aperture values of at least0.6, least 0.65, at least 0.7, at least 0.75, at least 0.8, at least0.85, at least 0.9, at least 0.95, at least 1.0, or higher. In someimplementations, the optical compensation optics (e.g., an opticalcompensator or compensator) comprises a refractive optical element suchas a lens, a plate of optically-transparent material such as glass, aplate of optically-transparent material such as glass, or in the case ofpolarized light beams, a quarter-wave plate or half-wave plate, etc..Other configurations may be employed to enable the first and secondsurfaces to be imaged at different times. For example, one or morelenses or optical elements may be configured to be translated in and outof, or along, an optical path between the objective lens 110 and theimage sensor.

In certain designs, however, the objective lens 110 is configured toprovide sufficiently large depth of focus and/or depth of field toenable the first and second surfaces to be imaged with comparableoptical resolution without such compensation optics moving into and outof an optical path in the imaging module, such as an optical pathbetween the objective lens and the image sensor or photodetector array.Similarly, in various designs, the objective lens 110 is configured toprovide sufficiently large depth of focus and/or depth of field toenable the first and second surfaces to be imaged with comparableoptical resolution without optics being moved, such as one or morelenses or other optical components being translated along an opticalpath in the imaging module, such as an optical path between theobjective lens and the image sensor or photodetector array. Examples ofsuch objective lenses will be described in more detail below.

In some implementations, the objective lens (or microscope objective)110 may be configured to have reduced magnification. The objective lens110 may be configured, for example, such that the fluorescence imagingmodule has a magnification of from less than 2x to less than 10× (aswill be discussed in more detail below). Such reduced magnification mayalter design constraints such that other design parameters can beachieved. For example, the objective lens 110 may also be configuredsuch that the fluorescence imaging module has a large field-of-view(FOV) ranging, for example, from about 1.0 mm to about 5.0 mm (e.g., indiameter, width, length, or longest dimension) as will be discussed inmore detail below.

In some implementations, the objective lens 110 may be configured toprovide the fluorescence imaging module with a field-of-view asindicated above such that the FOV has diffraction-limited performance,e.g., less than 0.15 waves of aberration over at least 60%, 70%, 80%,90%, or 95% of the field, as will be discussed in more detail below.

In some implementations, the objective lens 110 may be configured toprovide the fluorescence imaging module with a field-of-view asindicated above such that the FOV has diffraction-limited performance,e.g., a Strehl ratio of greater than 0.8 over at least 60%, 70%, 80%,90%, or 95% of the field, as will be discussed in more detail below.

Referring again to FIGS. 2A and 2B, the first dichroic beam splitter orbeam combiner is disposed in the first optical path between the lightsource and the sample so as to illuminate the sample with one or moreexcitation beams. This first dichroic beam splitter or combiner is alsoin one or more second optical path(s) from the sample to the differentoptical channels used to detect the fluorescence emission. Accordingly,the first dichroic filter 130 couples the first optical path of theexcitation beam emitted by the illumination source 115 and secondoptical path of the emission light emitted by a sample specimen to thevarious optical channels where the light is directed to respective imagesensors or photodetector arrays for capturing images of the sample.

In various implementations, the first dichroic filter 130, e.g., firstdichroic reflector or beam splitter or beam combiner, has a passbandselected to transmit light from the illumination source 115 only withina specified wavelength band or possibly a plurality of wavelength bandsthat include the desired excitation wavelength or wavelengths. Forexample, the first dichroic beam splitter 130 includes a reflectivesurface comprising a dichroic reflector that has spectral transmissivityresponse that is, e.g., configured to transmit light having at leastsome of the wavelengths output by the light source that form part of theexcitation beam. The spectral transmissivity response may be configurednot to transmit (e.g., instead to reflect) light of one or more otherwavelengths, for example, of one or more other fluorescence emissionwavelengths. In some implementations, the spectral transmissivityresponse may also be configured not to transmit (e.g., instead toreflect) light of one or more other wavelengths output by the lightsource. Accordingly, the first dichroic filter 130 may be utilized toselect which wavelength or wavelengths of light output by the lightsource reach the sample. Conversely, the dichroic reflector in the firstdichroic beam splitter 130 has a spectral reflectivity response thatreflects light having one or more wavelengths corresponding to thedesired fluorescence emission from the sample and possible reflectslight having one or more wavelengths output from the light source thatis not intended to reach the sample. Accordingly, in someimplementations, the dichroic reflector has a spectral transmissivitythat includes one or more pass bands to transmit the light to beincident on the sample and one or more stop bands that reflects lightoutside the pass bands, for example, light at one or more emissionwavelengths and possibly one or more wavelengths output by the lightsource that are not intended to reach the sample. Likewise, in someimplementations the dichroic reflector has a spectral reflectivity thatincludes one or more spectral regions configured to reflect one or moreemission wavelengths and possible one or more wavelengths output by thelight source that are not intended to reach the sample and includes oneor more regions that transmit light outside these reflection regions.The dichroic reflector included in the first dichroic filter 130 maycomprise a reflective filter such as an interference filter (e.g., aquarter-wave stack) configured to provide the appropriate spectraltransmission and reflection distributions. FIGS. 2A and 2B also show adichroic filter 105, which may comprise for example a dichroic beamsplitter or beam combiner, that may be used to direct the autofocuslaser 102 though the objective and to the sample support structure.

Although the imaging module 100 shown in FIGS. 2A and 2B and discussedabove is configured such that the excitation beam is transmitted by thefirst dichroic filter 130 to the objective lens 110, in some designs theillumination source 115 may be disposed with respect to the firstdichroic filter 130 and/or the first dichroic filter is configured(e.g., oriented) such that the excitation beam is reflected by the firstdichroic filter 130 to the objective lens 110. Similarly, in some suchdesigns, the first dichroic filter 130 is configured to transmitfluorescence emission from the sample and possibly transmit light havingone or more wavelengths output from the light source that is notintended to reach the sample. As will be discussed below, a design wherethe fluorescence emission is transmitted instead of reflected maypotentially reduce wavefront error in the detected emission and/orpossibly have other advantages. In either case, in variousimplementations the first dichroic reflector 130 is disposed in thesecond optical path so as to receive fluorescence emission from thesample, at least some of which continues on to the detection channels120.

FIGS. 3A and 3B illustrate the optical paths within the multi-channelfluorescence imaging module of FIGS. 2A and 2B. In the example show inFIG. 2A and FIG. 3A, the detection channels 120 are disposed to receivefluorescence emission from a sample specimen that is transmitted by theobjective lens 110 and reflected by the first dichroic filter 130. Asreferred to above and described more below, in some designs thedetection channels 120 may be disposed to receive the portion of theemission light that is transmitted, rather than reflected, by the firstdichroic filter. In either case, the detection channels 120 may includeoptics for receiving at least a portion of the emission light. Forexample, the detection channels 120 may include one or more lenses, suchas tube lenses, and may include one or more image sensors or detectorssuch as photodetector arrays (e.g., CCD or CMOS sensor arrays) forimaging or otherwise producing a signal based on the received light. Thetube lenses may, for example, comprise one or more lens elementsconfigured to form an image of the sample onto the sensor orphotodetector array to capture an image thereof. Additional discussionof detection channels is included below and in U.S. ProvisionalApplication No. 62/962,723, filed Jan. 17, 2020, which is incorporatedherein by reference in its entirety. In some instances, improved opticalresolution may be achieved using an image sensor having relatively highsensitivity, small pixels, and high pixel count, in conjunction with asuitable sampling scheme, which may include oversampling orundersampling.

FIGS. 3A and 3B are ray tracing diagrams illustrating optical paths ofthe illumination and imaging module 100 of FIGS. 2A and 2B. FIG. 3Acorresponds to a top view of the illumination and imaging module 100.FIG. 3B corresponds to a side view of the illumination and imagingmodule 100. The illumination and imaging module 100 illustrated in thesefigures includes four detection channels 120. However, it will beunderstood that the disclosed illumination and imaging modules mayequally be implemented in systems including more or fewer than fourdetection channels 120. For example, the multi-channel systems disclosedherein may be implemented with as few as one detection channel 120, oras many as two detection channels 120, three detection channels 120,four detection channels 120, five detection channels 120, six detectionchannels 120, seven detection channels 120, eight detection channels120, or more than eight detection channels 120, without departing fromthe spirit or scope of the present disclosure.

The non-limiting example of imaging module 100 illustrated in FIGS. 3Aand 3B includes four detection channels 120, a first dichroic filter 130that reflects a beam 150 of emission light, a second dichroic filter(e.g., a dichroic beam splitter) 135 that splits the beam 150 into atransmitted portion and a reflected portion, and two channel-specificdichroic filters (e.g., dichroic beam splitters) 140 that further splitthe transmitted and reflected portions of the beam 150 among individualdetection channels 120. The dichroic reflecting surface in the dichroicbeam splitters 135 and 140 for splitting the beam 150 among detectionchannels are shown disposed at 45 degrees relative to a central beamaxis of the beam 150 or an optical axis of the imaging module. However,as discussed below, an angle smaller than 45 degrees may be employed andmay offer advantages such as sharper transitions from pass band to stopband.

The different detection channels 120 includes imaging devices 124, whichmay include an image sensor or photodetector array (e.g., a CCD or CMOSdetector array). The different detection channels 120 further includeoptics 126 such as lenses (e.g., one or more tube lenses each comprisingone or more lens elements) disposed to focus the portion of the emissionlight entering the detection channel 120 at a focal plane coincidentwith a plane of the photodetector array 124. The optics 126 (e.g., atube lens) combined with the objective lens 110 are configured to forman image of the sample onto the photodetector array 124 to capture animage of the sample, for example, an image of a surface on the flow cellor other sample support structure after the sample has bound to thatsurface. Accordingly, such an image of the sample may comprise aplurality of fluorescent emitting spots or regions across a spatialextent of the sample support structure where the sample is emittingfluorescence light. The objective lens 110 together with the optics 126(e.g., tube lens) may provide a field of view (FOV) that includes aportion of the sample or the entire sample. Similarly, the photodetectorarray 124 of the different detection channels 120 may be configured tocapture images of a full field of view (FOV) provided by the objectivelens and the tube lens, or a portion thereof. In some implementations,the photodetector array 124 of some or all detection channels 120 candetect the emission light emitted by a sample disposed on the samplesupport structure, e.g., a surface of the flow cell, or a portionthereof and record electronic data representing an image thereof. Insome implementations, the photodetector array 124 of some or alldetection channels 120 can detect features in the emission light emittedby a specimen without capturing and/or storing an image of the sampledisposed on the flow cell surface and/or of the full field of view (FOV)provided by the objective lens and optics 126 and/or 122 (e.g., elementsof a tube lens). In some implementations, the FOV of the disclosedimaging modules (e.g., that provided by the combination of objectivelens 110 and optics 126 and/or 122) may range, for example, betweenabout 1 mm and 5 mm (e.g., in diameter, width, length, or longestdimension) as will be discussed below. The FOV may be selected, forexample, to provide a balance between magnification and resolution ofthe imaging module and/or based on one or more characteristics of theimage sensors and/or objective lenses. For example, a relatively smallerFOV may be provided in conjunction with a smaller and faster imagingsensor to achieve high throughput.

Referring again to FIGS. 3A and 3B, in some implementations, the optics126 in the detection channel (e.g., the tube lens) may be configured toreduce optical aberration in images acquired using optics 126 incombination with objective lens 110. In some implementations comprisingmultiple detection channels for imaging at different emissionwavelengths, the optics 126 (e.g., the tube lens) for differentdetection channels have different designs to reduce aberration for therespective emission wavelengths at which that particular channel isconfigured to image. In some implementations, the optics 126 (e.g., thetube lens) may be configured to reduce aberrations when imaging aspecific surface (e.g., a plane, object plane, etc.) on the samplesupport structure comprising fluorescing sample sites disposed thereonas compared to other locations (e.g., other planes in object space).Similarly, in some implementations, the optics 126 (e.g., the tube lens)may be configured to reduce aberrations when imaging first and secondsurfaces (e.g., first and second planes, first and second object planes,etc.) on a dual surface sample support structure (e.g., a dual surfaceflow cell) having fluorescing sample sites disposed thereon as comparedto other locations (e.g., other planes in object space). For example,the optics 126 in the detection channel (e.g., tube lens) may bedesigned to reduce the aberration at two depths or planes located atdifferent distances from the objective lens as compared to theaberrations associated with other depths or planes at other distancesfrom the objective. For example, optical aberration may be less forimaging the first and second surfaces than elsewhere in a region fromabout 1 to about 10 mm from the objective lens. Additionally, customoptic 126 in the detection channel (e.g., a tube lens) may in someembodiments be configured to compensate for aberration induced bytransmission of emission light through one or more portions of thesample support structure such as a layer that includes one of thesurfaces on which the sample is disposed as well as possibly a solutionadjacent to and in contact with the surface on which the sample isdisposed. The layer comprising one of the surfaces on which the sampleis disposed may comprise, e.g., glass, quartz, plastic, or othertransparent material having a refractive index, and which introducesoptical aberration. Custom optic 126 in the detection channel (e.g., thetube lens), for example, may in some implementations be configured tocompensate for optical aberration induced by a sample support structure,e.g., a coverslip or flow cell wall, or other sample support structurecomponents, as well as possibly a solution adjacent to and in contactwith the surface on which the sample is disposed.

In some implementations, the optics 126 in the detection channel (e.g.,a tube lens) are configured to have reduced magnification. The optics126 in the detection channel (e.g., a tube lens) may be configured, forexample, such that the fluorescence imaging module has a magnificationof less than, for example, 10×, as will be discussed further below. Suchreduced magnification may alter design constraints such that otherdesign parameters can be achieved. For example, the optics 126 (e.g., atube lens) may also be configured such that the fluorescence imagingmodule has a large field-of-view (FOV), for example, of at least 1.0 mmor larger (e.g., in diameter, width, length, or longest dimension), aswill be discussed further below.

In some implementations, the optics 126 (e.g., a tube lens) may beconfigured to provide the fluorescence imaging module with afield-of-view as indicated above such that the FOV has less than 0.15waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of thefield, as will be discussed further below.

Referring again to FIGS. 3A and 3B, in various implementations, a sampleis located at or near a focal position 112 of the objective lens 110. Asdescribed above with reference to FIGS. 2A and 2B, a light source suchas a laser source provides an excitation beam to the sample to inducefluorescence. At least a portion of fluorescence emission is collectedby the objective lens 110 as emission light. The objective lens 110transmits the emission light toward the first dichroic filter 130, whichreflects some or all of the emission light as the beam 150 incident uponthe second dichroic filter 135 and to the different detection channels,each comprising optics 126 that form an image of the sample (e.g., aplurality of fluorescing sample sites on a surface of a sample supportstructure) onto a photodetector array 124.

As discussed above, in some implementations, the sample supportstructure comprises a flow cell such as a dual surface flow cell havingtwo surfaces (e.g., two interior surfaces, a first surface and a secondsurface, etc.) containing sample sites that emit fluorescent emission.These two surfaces may be separated by a distance from each other in thelongitudinal (Z) direction along the direction of the central axis ofthe excitation beam and/or the optical axis of the objective lens. Thisseparation may correspond, for example, to a flow channel within theflow cell. Analytes or reagents may be flowed through the flow channeland contact the first and second interior surfaces of the flow cell,which may thereby be contacted with a binding composition such thatfluorescence emission is radiated from a plurality of sites on the firstand second interior surfaces. The imaging optics (e.g., objective lens110) may be positioned at a suitable distance (e.g., a distancecorresponding to the working distance) from the sample to form in-focusimages of the sample on one or more detector arrays 124. As discussedabove, in various designs, the objective lens 110 (possibly incombination with the optics 126) may have a depth of field and/or depthof focus that is at least as large as the longitudinal separationbetween the first and second surfaces. The objective lens 110 and theoptics 126 (of each detection channel) can thus simultaneously formimages of both the first and the second flow cell surfaces on thephotodetector array 124, and these images of the first and secondsurfaces are both in focus and have comparable optical resolution (ormay be brought into focus with only minor refocusing of the objects toacquire images of the first and second surfaces that have comparableoptical resolution). In various implementations, compensation opticsneed not be moved into or out of an optical path of the imaging module(e.g., into or out of the first and/or second optical paths) to formin-focus images of the first and second surfaces that are of comparableoptical resolution. Similarly, in various implementations, one or moreoptical elements (e.g., lens elements) in the imaging module (e.g., theobjective lens 110 or optics 126) need not be moved, for example, in thelongitudinal direction along the first and/or second optical paths toform in-focus images of the first surface in comparison to the locationof said one or more optical elements when used to form in-focus imagesof the second surface. In some implementations, the imaging moduleincludes an autofocus system configured to quickly and sequentiallyrefocus the imaging module on the first and/or second surface such thatthe images have comparable optical resolution. In some implementations,objective lens 110 and/or optics 126 are configured such that both thefirst and second flow cell surfaces are in focus simultaneously withcomparable optical resolution without moving an optical compensator intoor out of the first and/or second optical path, and without moving oneor more lens elements (e.g., objective lens 110 and/or optics 126 (suchas a tube lens) longitudinally along the first and/or second opticspath. In some implementations, images of the first and/or secondsurfaces, acquired either sequentially (e.g., with refocusing betweensurfaces) or simultaneously (e.g., without refocusing between surfaces)using the novel objective lens and/or tube lens designs disclosedherein, may be further processed using a suitable image processingalgorithm to enhance the effective optical resolution of the images suchthat the images of the first and second surfaces have comparable opticalresolution. In various implementations, the sample plane is sufficientlyin focus to resolve sample sites on the first and/or second flow cellsurfaces, the sample sites being closely spaced in lateral directions(e.g., in the X and Y directions).

As discussed above, the dichroic filters may comprise interferencefilters that selectively transmit and reflect light of differentwavelengths based on the principle of thin-film interference, usinglayers of optical coatings having different refractive indices andparticular thickness. Accordingly, the spectral response (e.g.,transmission and/or reflection spectra) of the dichroic filtersimplemented within multi-channel fluorescence imaging modules may be atleast partially dependent upon the angle of incidence, or range ofangles of incidence, at which the light of the excitation and/oremission beams are incident upon the dichroic filters. Such effects maybe especially significant with respect to the dichroic filters of thedetection optical path (e.g., the dichroic filters 135 and 140 of FIGS.3A and 3B).

FIG. 4 is a graph illustrating a relationship between dichroic filterperformance and beam angle of incidence (AOI). Specifically, the graphof FIG. 4 illustrates the effect of angle of incidence on the transitionwidth or spectral span of a dichroic filter, which corresponds to therange of wavelengths where the spectral response (e.g., transmissionspectrum and/or reflection spectrum) transitions between the passbandand stopband regions of a dichroic filter. Thus, a transmission edge (orreflection edge) having a relatively small spectral span (e.g., a smalldelta λ value in the graph of FIG. 4) corresponds to a sharpertransition between passband and stopband regions or the transmission andreflection regions (or conversely between reflection and transmissionregions), while a transmission edge (or reflection edge) having arelatively large spectral span (e.g., a large delta_λ value in the graphof FIG. 4) corresponds to a less sharp transition between passband andstopband regions. In various implementations, sharper transitionsbetween passband and stopband regions are generally desirable. Moreover,it may also be desirable to have increased consistency or a relativelyconsistent transition width across all or most of the field of viewand/or beam area.

Fluorescence imaging modules, in which the dichroic mirrors are disposedat 45 degrees relative to a central beam axis of the emission light orthe optical axis of the optical paths (e.g., of the objective lensand/or tube lens), accordingly can have a transition width of roughly 50nm for an example dichroic filter, as shown in FIG. 4. Because theemission light beam is not collimated and has some degree of divergence,fluorescence imaging modules may have a range of angles of incidence ofapproximately 5 degrees between opposing sides of the beam. Thus, asshown in FIG. 4, different portions of the beam of emission light may beincident upon a channel splitting dichroic filter at various angles ofincidence between 40 degrees and 50 degrees. This range of relativelylarge angles of incidence corresponds to a range of transition widthsbetween about 40 nm and about 62 nm. This range of relatively largeangles of incidence thereby leads to an increase in transition width ofthe dichroic filter in the imaging module. Performance of multi-channelfluorescence imaging modules may thus be improved by providing smallerangles of incidence across the full beam, thereby making thetransmission edge sharper and allowing better discrimination betweendifferent fluorescence emission bands.

FIG. 5 is a graph illustrating a relationship between beam footprintsize (DBS) and beam angle of incidence (DBS angle) on a dichroic filter.In some instances, a smaller beam footprint may be desirable. Forexample, a small beam footprint allows smaller dichroic filters to beused to split a beam into different wavelength ranges. The use ofsmaller dichroic filters in turn reduces manufacturing costs andimproves the ease of manufacturing suitably flat dichroic filters. Asshown in FIG. 5, any angle of incidence greater than 0 degrees (i.e.,perpendicular to the surface of the dichroic filter) results in anelliptical beam footprint having an area larger than the cross-sectionalarea of the beam. An angle of incidence of 45 degrees results in a largebeam footprint on the dichroic reflector that is greater than 1.4 timesthe cross-sectional area of the beam when incident at zero degrees.

FIGS. 6A and 6B schematically illustrate a non-limiting exampleconfiguration of dichroic filters and detection channels in amulti-channel fluorescence imaging module where the dichroic mirrors aredisposed at an angle of less than 45 degrees relative to a central beamaxis of the emission light or the optical axis of the optical paths(e.g., of the objective lens and/or tube lens). FIG. 6A depicts animaging module 500 including a plurality of detection channels 520 a,520 b, 520 c, 520 d. FIG. 6B is a detailed view of the portion of theimaging module 500 within the circle 5B as shown in FIG. 6A. As will bedescribed in greater detail, the configuration illustrated in FIGS. 6Aand 6B includes a number of aspects that may result in significantimprovements over conventional multi-channel fluorescence imaging moduledesigns. In some instances, fluorescence imaging modules and systems ofthe present disclosure may, however, may be implemented with one or asubset of the features described with respect to FIGS. 6A and 6B withoutdeparting from the spirit or scope of the present disclosure.

The imaging module 500 depicted in FIG. 6A includes an objective lens510 and four detection channels 520 a, 520 b, 520 c, and 520 d disposedto receive and/or image emission light transmitted by the objective lens510. A first dichroic filter 530 is provided to couple the excitationand detection optical paths. In contrast to the design shown in FIGS. 2Aand 2B, as well as in FIGS. 3A and 3B, the first dichroic filter 530(e.g., a dichroic beam splitter or combiner) is configured to reflectlight from the light source to the objective lens 510 and sample, andtransmit fluorescence emission from the sample to the detection channels520 a, 520 b, 520 c, and 520 d. A second dichroic filter 535 splits abeam of emission light among at least two detection channels 520 a, 520b by transmitting a first portion 550 a and reflecting a second portion550 b. Additional dichroic filters 540 a, 540 b are provided to furthersplit the emission light. Dichroic filter 540 a transmits at least aportion of the first portion 550 a of the emission light and reflects aportion 550 c to a third detection channel 520 c. Dichroic filter 540 btransmits at least a portion of the second portion 550 b of the emissionlight and reflects a portion 550 d to a fourth detection channel 520 d.Although the imaging module 500 is depicted with four detectionchannels, in various embodiments the imaging module 500 may include moreor fewer detection channels, with a correspondingly larger or smallernumber of dichroic filters as appropriate to provide a portion of theemission light to each detection channel. For example, in someembodiments, the features of the imaging module 500 may be implementedwith similar advantageous effects in a simplified imaging moduleincluding only two detection channels 520 a, 520 b, and omittingadditional dichroic filters 540 a, 540 b. In some implementations, onlyone detection channel may be included. Alternatively, three or moredetection channels may be employed.

The detection channels 520 a, 520 b, 520 c, 520 d illustrated in FIG. 6Amay include some or all of the same or similar components to those ofthe detection channels 120 illustrated in FIGS. 2A-3B. For example,different detection channel 520 a, 520 b, 520 c, 520 d may include oneor more image sensors or photodetectors arrays, and may includetransmissive and/or reflective optics such as one or more lenses (e.g.,tube lenses) that focus the light received by the detection channel ontoits respective image sensor or photodetector array.

The objective lens 510 is disposed to receive emission light emitted byfluorescence from a specimen. In particular, the first dichroic filter530 is disposed to receive the emission light collected and transmittedby the objective lens 510. As discussed above and shown in FIG. 6A, insome designs, an illumination source (e.g., the illumination source 115of FIGS. 2A and 2B) such as a laser source or the like is disposed toprovide an excitation beam which is incident on the first dichroicfilter 530 such that the first dichroic filter 530 reflects theexcitation beam into the same objective lens 510 that transmits theemission light, for example, in an epifluorescence configuration. Insome other designs, the illumination source may be directed to thespecimen by other optical components along a different optical path thatdoes not include the same objective lens 510. In such configurations,the first dichroic filter 530 may be omitted.

Similarly, as discussed above and shown in FIG. 6A, the detection optics(e.g., including the detection channels 520 a, 520 b, 520 c, 520 d andany optical components such as dichroic filters 535, 540 a, 540 b alongthe optical path between the objective lens 510 and the detectionchannels 520 a, 520 b, 520 c, 520 d) may be disposed on the transmissionpath of the first dichroic filter 530, rather than on the reflected pathof the first dichroic filter 530. In one example implementation, theobjective lens 510 and detection optics are disposed such that theobjective lens 510 transmits the beam 550 of emission light directlytoward the second dichroic filter 535. The wavefront quality of theemission light may be degraded somewhat by the presence of the firstdichroic filter 530 along the path of the beam 550 of emission light(e.g., by imparting some wavefront error to the beam 550). However, thewavefront error introduced by a beam transmitted through a dichroicreflector of a dichroic beam splitter is generally significantly smallerthan the wavefront error of a beam reflected from the dichroicreflecting surface of a dichroic beam splitter (e.g., an order ofmagnitude smaller). Thus, the wavefront quality and subsequent imagingquality of the emission light in a multi-channel fluorescence imagingmodule may be substantially improved by placing the detection opticsalong the transmitted beam path of the first dichroic filter 530 ratherthan along the reflected beam path.

Still referring to FIG. 6A, within the detection optics of the imagingmodule 500, dichroic filters 535, 540 a, and 540b are provided to splitthe beam 550 of emission light among the detection channels 520 a, 520b, 520 c, 520 d. For example, the dichroic filters 535, 540 a, and 540 bsplit the beam 550 on the basis of wavelength, such that a firstwavelength or wavelength band of the emission light can be received bythe first detection channel 520 a, a second wavelength or wavelengthband of the emission light can be received by the second detectionchannel 520 b, a third wavelength or wavelength band of the emissionlight can be received by the third detection channel 520 c, and a fourthwavelength or wavelength band of the emission light can be received bythe fourth detection channel 520 d. In some implementations, multipleseparated wavelengths or wavelength bands can be received by thedetection channel.

In contrast to the multi-channel fluorescence imaging module designshown in FIGS. 2A and 2B, as well as FIGS. 3A and 3B, the imaging module500 has dichroic filters 535, 540 a, and 540 b disposed at angles ofincidence of less than 45 degrees with respect to the central beam axisof the incident beams. As shown in FIG. 6B, the different beams 550, 550a, 550 b have respective central beam axes 552, 552 a, 552 b. In variousimplementations, the central beam axes 552, 552 a, 552 b is at thecenter of a cross-section of the beam orthogonal to the propagationdirection of the beam. These central beam axes 552, 552 a, 552 b maycorrespond to the optical axis of the objective lens and/or the opticswithin the separate channels, for example, the optical axes of therespective tube lenses. Additional rays 554, 554 a, 554 b of each beam550, 550 a, 550 b are illustrated in FIG. 6B to indicate the diameter ofeach beam 550, 550 a, 550 b. Beam diameter may be defined, for example,as a full width at half maximum diameter, a D46 (i.e., 4 times 6, where6 is the standard deviation of the horizontal or vertical marginaldistribution of the beam respectively) or second-moment width, or anyother suitable definition of beam diameter.

The central beam axis 552 of the beam 550 of emission light may serve asa reference point for defining the angle of incidence of the beam 550 onthe second dichroic filter 535. Accordingly, the “angle of incidence”(AOI) of a beam 550 may be the angle between the central beam axis 552of the incident beam 550 and a line N normal to the surface the beam isincident on, for example, the dichroic reflective surface. When the beam550 of emission light is incident upon the dichroic reflective surfaceof the second dichroic filter 535 at an angle of incidence AOI, thesecond dichroic filter 535 transmits a first portion 550 a of theemission light (e.g., the portion having wavelengths within the passbandregion of the second dichroic filter 535) and reflects a second portion550 b of the emission light (e.g., the portion having wavelengths withinthe stopband region of the second dichroic filter 535). The firstportion 550 a and the second portion 550 b may each be similarlydescribed in terms of a central beam axis 552 a, 552 b. As referred toabove, the optical axis may alternatively or additionally be used.

In the example configuration of FIGS. 6A and 6B, the second dichroicfilter 535 is disposed such that the central beam axis 552 of the beam550 is incident at an angle of incidence of 30 degrees. Similarly, theadditional dichroic filters 540 a, 540 b are disposed such that thecentral beam axes 552 a, 552 b of the first and second portions 550 a,550 b of the beam 550 are also incident at angles of incidence of 30degrees. However, in various implementations these angles of incidencemay be other angles smaller than 45 degrees. In some instances, forexample, the angles of incidence may range between about 20 degrees andabout 45 degrees, as will be discussed further below. Moreover, theangles of incidence on each of the dichroic filters 535, 540 a, 540 bneed not necessarily be the same. In some embodiments, some or all ofthe dichroic filters 535, 540 a, 540 b may be disposed such that theirincident beams 550, 550 a, 550 b have different angles of incidence. Asdescribed above, the angle of incidence may be with respect to theoptical axis of the optics within the imaging module, for example, theobjective lens and/or the optics in the detection channels (e.g., thetube lenses) and the dichroic reflective surface in the respectivedichroic beam splitter. The same ranges and values for the angle ofincidence apply to the case when the optical axis is used to specify theAOI.

The beams 550, 550 a, 550 b of emission light in a fluorescence imagingmodule system are typically diverging beams. As noted above, the beamsof emission light can have a beam divergence large enough that regionsof the beam within the beam diameter are incident upon the dichroicfilters at angles of incidence that differ by up to 5 degrees or morerelative to the angle of incidence of the central beam axis and/oroptical axis of the optics. In some designs, the objective lens 510 maybe configured, for example, to have an f-number or numerical apertureselected to produce a smaller beam diameter for a given field of view ofthe microscope. In one example, the f-number or numerical aperture ofthe objective lens 510 may be selected such that the full diameter ofthe beams 550, 550 a, 550 b are incident upon dichroic filters 535, 540a, 540 b at angles of incidence within, for example, 1 degree, 1.5degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5degrees, or 5 degrees of the angle of incidence of the central beam axes552, 552 a, 552 b.

In some implementations, the focal length of the objective lens that issuitable for producing such a narrow beam diameter may be longer thanthose typically employed in fluorescence microscopes or imaging systems.For example, in some implementations, the focal length of the objectivelens may range between 20 mm and 40 mm, as will be discussed furtherbelow. In one example, an objective lens 510 having a focal length of 36mm may produce a beam 550 characterized by a divergence small enoughthat light across the full diameter of the beam 550 is incident upon thesecond dichroic filter 535 at angles within 2.5 degrees of the angle ofincidence of the central beam axis.

FIG. 7 and FIG. 8 provide graphs illustrating improved dichroic filterperformance due to aspects of the imaging module configuration of FIGS.6A and 6B (or any of the imaging module configurations disclosedherein). The graph in FIG. 7 is similar to that of FIG. 4 andillustrates the effect of angle of incidence on the transition width(e.g., the spectral span of the transmission edge) of a dichroic filter.FIG. 7 shows an example where the orientation of a dichroic filter(e.g., dichroic filters 535, 540 a, and 540 b) and the dichroicreflective surface therein is such that its incident beam has an angleof incidence of 30 degrees, rather than 45 degrees. FIG. 7 shows howthis reduced angle of incidence significantly improves the sharpness andthe uniformity of the transition width across the full beam diameter.For example, while an angle of incidence of 45 degrees at the centralbeam axis results in a range of transition widths between about 40 nmand about 62 nm, an angle of incidence of 30 degrees at the central beamaxis results in a range of transition widths between about 16 nm andabout 30 nm. In this example, the average transition width is reducedfrom about 51 nm to about 23 nm, indicating a sharper transition betweenpassband and stopband. Moreover, the variation in transition widthsacross the beam diameter is reduced by nearly 40% from a 22 nm range toa 14 nm range, indicating a more uniform sharpness of the transitionover the area of the beam.

FIG. 8 illustrates additional advantages that may be realized byselecting the appropriate f-number or numerical aperture for theobjective lens to reduce beam divergence in any of the imaging moduleconfigurations disclosed herein. In some implementations, a longer focallength is used. In the example of FIG. 8, the objective lens 510 has afocal length of 36 mm, which with the appropriate numerical aperture(e.g., less than 5), reduces the range of angles of incidence within thebeam 550 from 30 degrees ±5 degrees to 30 degrees ±2.5 degrees. Withthis design, the range of transition widths may be reduced to betweenabout 19 nm and about 26 nm. When compared to the improved system ofFIG. 7, although the average transition width is substantially the same(e.g., a spectral span of roughly 23 nm), the variation in transitionwidths across the beam diameter is further reduced to a 7 nm range,representing a reduction of nearly 70% relative to the range oftransition widths illustrated in FIG. 4.

Referring again to FIG. 5, the reduction in angle of incidence from 45degrees to 30 degrees at the central beam axis is further advantageousbecause it reduces the beam spot size on the dichroic filter. As shownin FIG. 5, an angle of incidence of 45 degrees results in a beamfootprint on the dichroic filter having an area greater than 1.4 timesthe cross-sectional area of the beam. However, an angle of incidence of30 degrees results in a beam footprint on the dichroic filter having anarea only about 1.15 times the cross-sectional area of the beam. Thus,reducing the angle of incidence at the dichroic filters 535, 540 a, 540b from 45 degrees to 30 degrees results in a reduction of about 18% inthe area of the beam footprint on the dichroic filters 535, 540 a, 540b. This reduction in beam footprint area allows smaller dichroic filtersto be used.

Referring now jointly to FIGS. 9A-B, the reduction in angle of incidencefrom 45 degrees to 30 degrees may also provide improved performance withregard to surface deformation caused by the dichroic filters in any ofthe imaging module configurations disclosed herein, as indicated byimprovements in the modulation transfer function. In general, the amountof surface deformation increases with larger area optical elements. If alarger area on the dichroic filter is employed, a larger amount ofsurface deformation is encountered, thereby introducing more wavefronterror into the beam. FIG. 9A illustrates the effect of folding angle onimage quality degradation induced by the addition of 1 wave ofpeak-to-valley (PV) spherical power to the last mirror. FIG. 9Billustrates the effect of folding angle on image quality degradationinduced by the addition of 0.1 wave of PV spherical power to the lastmirror. As shown in FIGS. 9A and 9B, the reduction in angle of incidenceto 30 degrees significantly reduces the effect of surface deformation toachieve close to diffraction-limited performance of the detectionoptics.

In some implementations of the disclosed imaging modules, thepolarization state of the excitation beam may be utilized to furtherimprove the performance of the multi-channel fluorescence imagingmodules disclosed herein. Referring back to FIGS. 2A, 2B, and 6A, forexample, some implementations of the multi-channel fluorescence imagingmodules disclosed herein have an epifluorescence configuration in whicha first dichroic filter 130 or 530 merges the optical paths of theexcitation beam and the beam of emission light such that both theexcitation and emission light are transmitted through the objective lens110, 510. As discussed above, the illumination source 115 may include alight source such as a laser or other source which provides the lightthat forms the excitation beam. In some designs, the light sourcecomprises a linearly polarized light source and the excitation beam maybe linearly polarized. In some designs, polarization optics are includedto polarize the light and/or rotate the polarization of the light. Forexample, a polarizer such as a linear polarizer may be included in anoptical path of the excitation beam to polarize the excitation beam.Retarders such as half wave retarders or a plurality of quarter waveretarders or retarders having other amounts of retardance may beincluded to rotate the linear polarization in some designs.

The linearly polarized excitation beam, when it is incident upon anydichroic filter or other planar interface, may be p-polarized (e.g.,having an electric field component parallel to the plane of incidence),s-polarized (e.g., having an electric field component normal to theplane of incidence), or may have a combination of p-polarization ands-polarization states within the beam. The p- or s-polarization state ofthe excitation beam may be selected and/or changed by selecting theorientation of the illumination source 115 and/or one or more componentsthereof with respect to the first dichroic filter 130, 530 and/or withrespect to any other surfaces with which the excitation beam willinteract. In some implementations where the light source output linearlypolarized light, the light source can be configured to provides-polarized light. For example, the light source may comprise an emittersuch as a solid-state laser or a laser diode that may be rotated aboutits optical axis or the central axis of the beam to orient the linearlypolarized light output therefrom. Alternatively, or in addition,retarders may be employed to rotate the linear polarization about theoptical axis or the central axis of the beam. As discussed above, insome implementations, for example when the light source does not outputpolarized light, a polarizer disposed in the optical path of theexcitation beam can polarize the excitation beam. In some designs, forexample, a linear polarizer is disposed in the optical path of theexcitation beam. This polarizer may be rotated to provide the properorientation of the linear polarization to provide s-polarized light.

In some designs, the linear polarization is rotated about the opticalaxis or the central axis of the beam such that s-polarization isincident on the dichroic reflector of the dichroic beam splitter. Whens-polarized light is incident on the dichroic reflector of the dichroicbeam splitter the transition between the pass band and the stop band issharper as opposed to when p-polarized light is incident on the dichroicreflector of the dichroic beam splitter.

As shown in FIGS. 10A and 10B, use of the p- or s-polarization state ofthe excitation beam may significantly affect the narrowband performanceof any excitation filters such as the first dichroic filter 130, 530.FIG. 10A illustrates a transmission spectrum between 610 nm and 670 nmfor an example bandpass dichroic filter at angles of incidence of 40degrees and 45 degrees, where the incident beam is linearly polarizedand is p-polarized with respect to the plane of the dichroic filter. Asshown in FIG. 10B, changing the orientation of the light source withrespect to the dichroic filter, such that the incident beam iss-polarized with respect to the plane of the dichroic filter, results ina substantially sharper edge between the passband and the stopband ofthe dichroic filter. Thus, the illumination and imaging modules 100, 500disclosed herein may advantageously have an illumination source 115oriented relative to the first dichroic filter 130, 530 such that theexcitation beam is s-polarized with respect to the plane of the firstdichroic filter 130, 530. As discussed above, in some implementation, apolarizer such as a linear polarizer may be used to polarize theexcitation beam. This polarizer may be rotated to provide an orientationof the linearly polarized light corresponding to s-polarized light. Alsoas discussed above, in some implementations, other approaches torotating the linearly polarized light may be used. For example, opticalretarders such as half wave retarders or multiple quarter wave retardersmay be used to rotate the polarization direction. Other arrangements arealso possible.

As discussed elsewhere herein, reducing the numerical aperture (NA) ofthe fluorescence imaging module and/or of the objective lens mayincrease the depth of field to enable the comparable imaging of the twosurfaces. FIGS. 11A-16B, show how the MTF is more similar at first andsecond surfaces separated by 1 mm of glass for lower numerical aperturesthan for larger numerical apertures.

FIGS. 11A and 11B show the MTF at first (FIG. 11A) and second (FIG. 11B)surfaces for an NA of 0.3.

FIGS. 12A and 12B show the MTF at first (FIG. 12A) and second (FIG. 12B)surfaces for an NA of 0.4.

FIGS. 13A and 13B show the MTF at first (FIG. 13A) and second (FIG. 13B)surfaces for an NA of 0.5.

FIGS. 14A and 14B show the MTF at first (FIG. 14A) and second (FIG. 14B)surfaces for an NA of 0.6.

FIGS. 15A and 15B show the MTF at first (FIG. 15A) and second (FIG. 15B)surfaces for an NA of 0.7.

FIGS. 16A and 16B show the MTF at first (FIG. 16A) and second (FIG. 16B)surfaces for an NA of 0.8. The first and second surfaces in each ofthese figures correspond to, e.g., the top and bottom surfaces of a flowcell.

FIGS. 17A-B provide plots of the calculated Strehl ratio (i.e., theratio of peak light intensity focused or collected by the optical systemversus that focused or collected by an ideal optical system and pointlight source) for imaging a second flow cell surface through a firstflow cell surface. FIG. 17A shows a plot of the Strehl ratios forimaging a second flow cell surface through a first flow cell surface asa function of the thickness of the intervening fluid layer (fluidchannel height) for different objective lens and/or optical systemnumerical apertures. As shown, the Strehl ratio decreases withincreasing separation between the first and second surfaces. One of thesurfaces would thus have deteriorated image quality with increasingseparation between the two surfaces. The decrease in second surfaceimaging performance with increased separation distance between the twosurfaces is reduced for imaging systems having smaller numeral aperturesas compared to those having larger numerical apertures. FIG. 17B shows aplot of the Strehl ratio as a function of numerical aperture for imaginga second flow cell surface through a first flow cell surface and anintervening layer of water having a thickness of 0.1 mm. The loss ofimaging performance at higher numerical apertures may be attributed tothe increased optical aberration induced by the fluid for the secondsurface imaging. With increasing NA, the increased optical aberrationintroduced by the fluid for the second surface imaging degrades theimage quality significantly. In general, however, reducing the numeralaperture of the optical system reduces the achievable resolution. Thisloss of image quality can be at least partially offset by providing anincreased sample plane (or object plane) contrast-to-noise ratio, forexample, by using chemistries for nucleic acid sequencing applicationsthat enhance the fluorescence emission for labeled nucleic acid clustersand/or that reduce background fluorescence emission. In some instances,for example, sample support structures comprising hydrophilic substratematerials and/or hydrophilic coatings may be employed. In some cases,such hydrophilic substrates and/or hydrophilic coatings may reducebackground noise. Additional discussion of sample support structures,hydrophilic surfaces and coatings, and methods for enhancingcontrast-to-noise ratios, e.g., for nucleic acid sequencingapplications, can be found below.

In some implementations, any one or more of the fluorescence imagingsystem, the illumination and imaging module 100, the imaging optics(e.g., optics 126), the objective lens, and/or the tube lens isconfigured to have reduced magnification, such as a magnification ofless than 10×, as will be discussed further below. Such reducedmagnification may adjust design constraints such that other designparameters can be achieved. For example, any one or more of thefluorescence microscope, illumination and imaging module 100, theimaging optics (e.g., optics 126), the objective lens or the tube lensmay also be configured such that the fluorescence imaging module has alarge field-of-view (FOV), for example, a field-of-view of at least 3.0mm or larger (e.g., in diameter, width, height, or longest dimension),as will be discussed further below. Any one or more of the fluorescenceimaging system, the illumination and imaging module 100, the imagingoptics (e.g., optics 126), the objective lens and/or the tube lens maybe configured to provide the fluorescence microscope with such afield-of-view such that the FOV has less than, e.g., 0.1 waves ofaberration over at least 80% of field. Similarly, any one or more of thefluorescence imaging system, illumination and imaging module 100, theimaging optics (e.g., optics 126), the objective lens and/or the tubelens may be configured such that the fluorescence imaging module hassuch a FOV and is diffraction limited or is diffraction limited oversuch an FOV.

As discussed above, in various implementations, a large field-of-view(FOV) is provided by the disclosed optical systems. In someimplementations, obtaining an increased FOV is facilitated in part bythe use of larger image sensors or photodetector arrays. Thephotodetector array, for example, may have an active area with adiagonal of at least 15 mm or larger, as will be discussed furtherbelow. As discussed above, in some implementations the disclosed opticalimaging systems provide a reduced magnification, for example, of lessthan 10× which may facilitate large FOV designs. Despite the reducedmagnification, the optical resolution of the imaging module may still besufficient as detector arrays having small pixel size or pitch may beused. The pixel size and/or pitch may, for example, be about 5 μm orless, as will be discussed in more detail below. In someimplementations, the pixel size is smaller than twice the opticalresolution provided by the optical imaging system (e.g., objective andtube lens) to satisfy the Nyquist theorem. Accordingly, the pixeldimension and/or pitch for the image sensor(s) may be such that aspatial sampling frequency for the imaging module is at least twice anoptical resolution of the imaging module. For example, the spatialsampling frequency for the photodetector array may be is at least 2times, at least 2.5 times, at least 3 times, at least 4 times, or atleast 5 times the optical resolution of the fluorescence imaging module(e.g., the illumination and imaging module, the objective and tube lens,the object lens and optics 126 in the detection channel, the imagingoptics between the sample support structure or stage configured tosupport the sample support stage and the photodetector array) or anyspatial sampling frequency in a range between any of these values.

Although a wide range of features are discussed herein with respect tofluorescence imaging modules, any of the features and designs describeherein may be applied to other types of optical imaging systemsincluding without limitation bright-field and dark-field imaging, andmay apply to luminescence or phosphorescence imaging.

Dual wavelength excitation/four channel imaging system: FIG. 18illustrates a dual excitation wavelength/four channel imaging system fordual-side imaging applications that includes an objective and tube lenscombination that is scanned in a direction perpendicular to the opticalaxis to provide for large area imaging, e.g., by tiling several imagesto create a composite image having a total field-of-view (FOV) that ismuch larger than that for each individual image. The system comprisestwo excitation light sources, e.g., lasers or laser diodes, operating atdifferent wavelengths and an autofocus laser. The two excitation lightbeams and autofocus laser beam are combined using a series of mirrorsand/or dichroic reflectors and delivered to an upper or lower interiorsurface of the flow cell through the objective. Fluorescence that isemitted by labeled oligonucleotides (or other biomolecules) tethered toone of the flow cell surfaces is collected by the objective, transmittedthrough the tube lens, and directed to one of four imaging sensorsaccording to the wavelength of the emitted light by a series ofintermediate dichroic reflectors. Autofocus laser light that has beenreflected from the flow cell surface is collected by the objective,transmitted through the tube lens, and directed to an autofocus sensorby a series of intermediate dichroic reflectors. The system allowsaccurate focus to be maintained (e.g., by adjusting the relativedistance between the flow cell surface and the objective using aprecision linear actuator, translation stage, or microscopeturret-mounted focus adjustment mechanism, to reduce or minimize thereflected light spot size on the autofocus image sensor) while theobjective/tube lens combination is scanned in a direction perpendicularto the optical axis of the objective. Dual wavelength excitation used incombination with four channel (i.e. four wavelength) imaging capabilityprovides for high-throughput imaging of the upper (near) and lower (far)interior surfaces of the flow cell.

Multiplexed optical read-heads:

In some instances, miniaturized versions of any of the imaging modulesdescribed herein may be assembled to create a multiplexed read-head thatmay be translated in one or more directions horizontally relative to asample surface, e.g., an interior surface of a flow cell, to imageseveral sections of the surface simultaneously. A non-limiting exampleof a multiplexed read-head has recently been described in U.S. PublishedPatent Application No. 2020/0139375 A1.

In some instances, for example, a miniaturized imaging module maycomprise a “microfluorometer” comprising an illumination or excitationlight source such as an LED or laser diode (or the tip of an opticalfiber connected to an external light source), one or more lenses forcollimating or focusing the illumination or excitation light, one ormore dichroic reflectors, one or more optical filters, one or moremirrors, beam-splitters, prisms, apertures, etc., one or moreobjectives, one or more custom tube lenses for enabling dual surfaceimaging with minimal focus adjustment as described elsewhere herein, oneor more image sensors, or any combination thereof, as describedelsewhere herein. In some instances, a miniaturized imaging module(e.g., a “microfluorometer”) may further comprise an autofocusmechanism, a microprocessor, power and data transfer connectors, alight-tight housing, etc. The resulting miniaturized imaging module maythus comprise an integrated imaging package or unit having a small formfactor. In some instances, the shortest dimension (e.g., width ordiameter) of the miniaturized imaging module may be less than 5 cm, lessthan 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than1.4 cm, less than 1.2 cm, less than 1 cm, less than 0.8 cm, or less than0.6 cm. In some instances, the longest dimension (e.g., height orlength) of the miniaturized imaging module may be less than 16 cm, lessthan 14 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than8 cm, less than 7 cm, less than 5 cm, less than 5 cm, less than 4.5 cm,less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, lessthan 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, lessthan 1.2 cm, or less than 1 cm. In some instances, one or moreindividual miniaturized imaging modules within the multiplexed read-headmay comprise an autofocus mechanism.

In some instances, multiplexed read-heads as described herein maycomprise an assembly of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than12 miniaturized imaging modules or microfluorometers held in fixedposition relative to each other. In some instances, the optical designspecifications and performance properties of the individual miniaturizedimaging modules or microfluorometers, e.g, for numerical aperture,field-of-view, depth-of-field, image resolution, etc., may be the sameas described elsewhere herein for other versions of the disclosedimaging modules. In some instances, the plurality of individualminiaturized imaging modules may be arranged in a linear arrangementcomprising one, two, three, four, or more than four rows and/or columns.In some instances, the plurality of individual miniaturized imagingmodules may be arranged in, e.g., a hexagonal close pack arrangement. Insome instances, the plurality of individual miniaturized imaging modulesmay be arranged in a circular or spiral arrangement, a randomlydistributed arrangement, or in any other arrangement known to those ofskill in the art.

FIGS. 43A-B provide non-limiting schematic illustrations of amultiplexed read-head as disclosed herein. FIG. 43A shows a side view ofa multiplexed read-head in which two rows of individualmicrofluorometers (seen from the end on) having common optical designspecifications, e.g., numerical aperture, field-of-view, workingdistance, etc., are configured to image a common surface, e.g., a firstinterior surface of a flow cell. FIG. 43B shows a top view of the samemultiplexed read-head illustrating the overlapping imaging pathsacquired by individual microfluorometers of the multiplexed read-head asthe read-head is translated relative to the flow cell (or vice versa).In some instances, the individual fields-of-view for the individualmicrofluorometers may overlap, as indicated in FIG. 43B. In someinstances, they may not overlap. In some instances, the multiplexed-readhead may be designed such that it aligns with and images predeterminedfeatures, e.g., individual fluid channels, within a flow cell.

FIGS. 44A-B provide non-limiting schematic illustrations of amultiplexed read-head where a first subset of the plurality ofindividual miniaturized imaging modules is configured to image a firstsample plane, e.g., a first interior surface of a flow cell, and asecond subset of the plurality is configured to simultaneously image asecond sample plane, e.g., a second interior surface of a flow cell.FIG. 44A shows a side view of the multiplexed read-head in which thefirst subset of individual microfluorimeters is configured to image,e.g., the first or upper interior surface of a flow cell, and the secondsubset is configured to image a second surface, e.g., the second orlower interior surface of a flow cell. FIG. 44B shows a top view of themultiplexed read-head of FIG. 44A illustrating the imaging pathsacquired by individual microfluorimeters of the multiplexed read-head.Again, in some instances, the individual fields-of-view for theindividual microfluorometers in a given subset may overlap. In someinstances, they may not overlap. In some instances, the multiplexed-readhead may be designed such that the individual miniaturized imagingmodules of the first and second subsets align with and imagepredetermined features, e.g., individual fluid channels, within a flowcell.

Improved or optimized objective and/or tube lens for use with thickercoverslips: Existing design practice includes the design of objectivelenses and/or use of commonly available off-the-shelf microscopeobjectives to optimize image quality when images are acquired throughthin (e.g., <200 μm thick) microscope coverslips. When used to image onboth sides of a fluidic channel or flow cell, the extra height of thegap between the two surfaces (i.e., the height of the fluid channel;typically, about 50 μm to 200 μm) introduces optical aberration inimages captured for the non-optimal side of the fluidic channel, therebycausing lower optical resolution. This is primarily because theadditional gap height is significant compared to the optimal coverslipthickness (typical fluid channel or gap heights of 50-200 μm vs.coverslip thicknesses of <200 μm). Another common design practice is toutilize an additional “compensator” lens in the optical path whenimaging is to be performed on the non-optimal side of the fluid channelor flow cell. This “compensator” lens and the mechanism required to moveit in or out of the optical path so that either side of the flow cellmay be imaged further increases system complexity and imaging systemdown time, and potentially degrades image quality due to vibration, etc.

In the present disclosure, the imaging system is designed forcompatibility with flow cell consumables that comprise a thickercoverslip or flow cell wall (thickness >700 μm). The objective lensdesign may be improved or optimized for a coverslip that is equal to thetrue cover slip thickness plus half of the effective gap thickness(e.g., 700 μm+1/2* fluid channel (gap) height). This designsignificantly reduces the effect of gap height on image quality for thetwo surfaces of the fluid channel and balances the optical quality forimages of the two surfaces, as the gap height is small relative to thetotal coverslip thickness and thus its impact on optical quality isreduced.

Additional advantages of using a thicker coverslip include improvedcontrol of thickness tolerance error during manufacturing, and a reducedlikelihood that the coverslip undergoes deformation due to thermal andmounting-induced stress. Coverslip thickness error and deformationadversely impact imaging quality for both the top surface and the bottomsurface of a flow cell.

To further improve the dual surface imaging quality for sequencingapplications, our optical system design places a strong emphasis onimproving or optimizing MTF (e.g., through improving or optimizing theobjective lens and/or tube lens design) in the mid- to high-spatialfrequency range that is most suitable for imaging and resolving smallspots or clusters.

Improved or optimized tube lens design for use in combination withcommercially available, off-the-shelf objectives: For low-cost sequencerdesign, the use of a commercially available, off-the-shelf objectivelens may be preferred due to its relatively low price. However, as notedabove, low-cost, off-the-shelf objectives are mostly optimized for usewith thin coverslips of about 170 μm in thickness. In some instances,the disclosed optical systems may utilize a tube lens design thatcompensates for a thicker flow cell coverslip while enabling high imagequality for both interior surfaces of a flow cell in dual-surfaceimaging applications. In some instances, the tube lens designs disclosedherein enable high quality imaging for both interior surfaces of a flowcell without moving an optical compensator into or out of the opticalpath between the flow cell and an image sensor, without moving one ormore optical elements or components of the tube lens along the opticalpath, and without moving one or more optical elements or components ofthe tube lens into or out of the optical path.

FIG. 19 provides an optical ray tracing diagram for a low lightobjective lens design that has been improved or optimized for imaging asurface on the opposite side of a 0.17 mm thick coverslip. The plot ofmodulation transfer function for this objective, shown in FIG. 20,indicates near-diffraction limited imaging performance when used withthe designed-for 0.17 mm thick coverslip.

FIG. 21 provides a plot of the modulation transfer function for the sameobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface on the opposite side of a 0.3 mm thickcoverslip. The relatively minor deviations of MTF value over the spatialfrequency range of about 100 to about 800 lines/mm (or cycles/mm)indicates that the image quality obtained even when using a 0.3 mm thickcoverslip is still reasonable.

FIG. 22 provides a plot of the modulation transfer function for the sameobjective lens illustrated in FIG. 19 as a function of spatial frequencywhen used to image a surface that is separated from that on the oppositeside of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueousfluid (i.e., under the kind of conditions encountered for dual-sideimaging of a flow cell when imaging the far surface). As can be seen inthe plot of FIG. 22, imaging performance is degraded, as indicated bythe deviations of the MTF curves from those for the an ideal,diffraction-limited case over the spatial frequency range of about 50lp/mm to about 900 lp/mm.

FIG. 23 and FIG. 24 provide plots of the modulation transfer function asa function of spatial frequency for the upper (or near) interior surface(FIG. 23) and lower (or far) interior surface (FIG. 24) of a flow cellwhen imaged using the objective lens illustrated in FIG. 19 through a1.0 mm thick coverslip, and when the upper and lower interior surfacesare separated by a 0.1 mm thick layer of aqueous fluid. As can be seen,imaging performance is significantly degraded for both surfaces.

FIG. 25 provides a ray tracing diagram for a tube lens design which, ifused in conjunction with the objective lens illustrated in FIG. 19,provides for improved dual-side imaging through a 1 mm thick coverslip.The optical design 700 comprising a compound objective (lens elements702, 703, 704, 705, 706, 707, 708, 709, and 710) and a tube lens (lenselements 711, 712, 713, and 714) is improved or optimized for use withflow cells comprising a thick coverslip (or wall), e.g., greater than700 μm thick, and a fluid channel thickness of at least 50 μm, andtransfers the image of an interior surface from the flow cell 701 to theimage sensor 715 with dramatically improved optical image quality andhigher CNR.

In some instances, the tube lens (or tube lens assembly) may comprise atleast two optical lens elements, at least three optical lens elements,at least four optical lens elements, at least five optical lenselements, at least six optical lens elements, at least seven opticallens elements, at least eight optical lens elements, at least nineoptical lens elements, at least ten optical lens elements, or more,where the number of optical lens elements, the surface geometry of eachelement, and the order in which they are placed in the assembly isimproved or optimized to correct for optical aberrations induced by thethick wall of the flow cell, and in some instances, allows one to use acommercially-available, off-the-shelf objective while still maintaininghigh-quality, dual-side imaging capability.

In some instances, as illustrated in FIG. 25, the tube lens assembly maycomprise, in order, a first asymmetric convex-convex lens 711, a secondconvex-plano lens 712, a third asymmetric concave-concave lens 713, anda fourth asymmetric convex-concave lens 714.

FIG. 26 and FIG. 27 provide plots of the modulation transfer function asa function of spatial frequency for the upper (or near) interior surface(FIG. 26) and lower (or far) interior surface (FIG. 27) of a flow cellwhen imaged using the objective lens (corrected for a 0.17 mm coverslip)and tube lens combination illustrated in FIG. 25 through a 1.0 mm thickcoverslip, and when the upper and lower interior surfaces are separatedby a 0.1 mm thick layer of aqueous fluid. As can be seen, the imagingperformance achieved is nearly that expected for a diffraction-limitedoptical design.

FIG. 28 provides ray tracing diagrams for tube lens design (left) of thepresent disclosure that has been improved or optimized to providehigh-quality, dual-side imaging performance. Because the tube lens is nolonger infinity-corrected, an appropriately designed null lens (right)may be used in combination with the tube lens to compensate for thenon-infinity-corrected tube lens for manufacturing and testing purposes.

Imaging channel-specific tube lens adaptation or optimization: Inimaging system design, it is possible to improve or optimize both theobjective lens and the tube lens in the same wavelength region for allimaging channels. Typically, the same objective lens is shared by allimaging channels (see, for example, FIG. 18), and each imaging channeleither uses the same tube lens or has a tube lens that shares the samedesign.

In some instances, the imaging systems disclosed herein may furthercomprise a tube lens for each imaging channel where the tube lens hasbeen independently improved or optimized for the specific imagingchannel to improve image quality, e.g., to reduce or minimize distortionand field curvature, and improve depth-of-field (DOF) performance foreach channel. Because the wavelength range (or bandpass) for eachspecific imaging channel is much narrower than the combined wavelengthrange for all channels, the wavelength- or channel-specific adaptationor optimization of the tube lens used in the disclosed systems resultsin significant improvements in imaging quality and performance. Thischannel-specific adaptation or optimization results in improved imagequality for both the top and bottom surfaces of the flow cell indual-side imaging applications.

Dual-side imaging w/o fluid present inflow cell: For optimal imagingperformance of both top and bottom interior surfaces of a flow cell, amotion-actuated compensator is typically required to correct for opticalaberrations induced by the fluid in the flow cell (typically comprisinga fluid layer thickness of about 50-200 μm). In some instances of thedisclosed optical system designs, the top interior surface of the flowcell may be imaged with fluid present in the flow cell. Once thesequencing chemistry cycle has been completed, the fluid may beextracted from the flow cell for imaging of the bottom interior surface.Thus, in some instances, even without the use of a compensator, theimage quality for the bottom surface is maintained.

Compensation for optical aberration and/or vibration usingelectro-optical phase plates: In some instances, dual-surface imagequality may be improved without requiring the removal of the fluid fromthe flow cell by using an electro-optical phase plate (or othercorrective lens) in combination with the objective to cancel the opticalaberrations induced by the presence of the fluid. In some instances, theuse of an electro-optical phase plate (or lens) may be used to removethe effects of vibration arising from the mechanical motion of amotion-actuated compensator and may provide faster image acquisitiontimes and sequencing cycle times for genomic sequencing applications.

Improved contrast-to-noise ratio (CNR), field-of-view (FOV), spectralseparation, and timing design to increase or maximize informationtransfer and throughput: Another way to increase or maximize informationtransfer in imaging systems designed for genomics applications is toincrease the size of the field-of-view (FOV) and reduce the timerequired to image a specific FOV. With typical large NA optical imagingsystems, it may be common to acquire images for fields-of-view that areon the order of 1 mm² in area, where in the presently disclosed imagingsystem designs large FOV objectives with long working distances arespecified to enable imaging of areas of 2 mm² or larger.

In some cases, the disclosed imaging systems are designed for use incombination with proprietary low-binding substrate surfaces and DNAamplification processes that reduce fluorescence background arising froma variety of confounding signals including, but are not limited to,nonspecific adsorption of fluorescent dyes to substrate surfaces,nonspecific nucleic acid amplification products (e.g., nucleic acidamplification products that arise the substrate surface in areas betweenthe spots or features corresponding to clonally-amplified clusters ofnucleic acid molecules (i.e., specifically amplified colonies),nonspecific nucleic acid amplification products that may arise withinthe amplified colonies, phased and pre-phased nucleic acid strands, etc.The use of low-binding substrate surfaces and DNA amplificationprocesses that reduce fluorescence background in combination with thedisclosed optical imaging systems may significantly cut down on the timerequired to image each FOV.

The presently disclosed system designs may further reduce the requiredimaging time through imaging sequence improvement or optimization wheremultiple channels of fluorescence images are acquired simultaneously orwith overlapping timing, and where spectral separation of thefluorescence signals is designed to reduce cross-talks betweenfluorescence detection channels and between the excitation light and thefluorescence signal(s).

The presently disclosed system designs may further reduce the requiredimaging time through improvement or optimization of scanning motionsequence. In the typical approach, an X-Y translation stage is used tomove the target FOV into position underneath the objective, an autofocusstep is performed where optimal focal position is determined and theobjective is moved in the Z direction to the determined focal position,and an image is acquired. A sequence of fluorescence images is acquiredby cycling through a series of target FOV positions. From an informationtransfer duty cycle perspective, information is only transferred duringthe fluorescence image acquisition portion of the cycle. In thepresently disclosed imaging system designs, a single-step motion inwhich all axes (X-Y-Z) are repositioned simultaneously is performed, andthe autofocus step is used to check focal position error. The additionalZ motion is only commanded if the focal position error (i.e., thedifference between the focal plane position and the sample planeposition) exceeds a certain limit (e.g., a specified error threshold).Coupled with high speed X-Y motion, this approach increases the dutycycle of the system, and thus increases the imaging throughput per unittime.

Furthermore, by matching the optical collection efficiency, modulationtransfer function, and image sensor performance characteristics of thedesign with the fluorescence photon flux expected for the inputexcitation photon flux, dye efficiency (related to dye extinctioncoefficient and fluorescence quantum yield), while accounting forbackground signal and system noise characteristics, the time required toacquire high quality (high contrast-to-noise ratio (CNR) images) may bereduced or minimized.

The combination of efficient image acquisition and improved or optimizedtranslation stage step and settle times leads to fast imaging times(i.e., the overall time required per field-of-view) and higherthroughput imaging system performance.

Along with the large FOV and fast image acquisition duty cycle, thedisclosed designs may comprise also specifying image plane flatness,chromatic focus performance between fluorescence detection channels,sensor flatness, image distortion, and focus quality specifications.

Chromatic focus performance is further improved by individually aligningthe image sensors for different fluorescence detection channels suchthat the best focal plane for each detection channel overlaps. Thedesign goal is to ensure that images across more than 90 percent of thefield-of-view are acquired within ±100 nm (or less) relative to the bestfocal plane for each channel, thus increasing or maximizing the transferof individual spot intensity signals. In some instances, the discloseddesigns further ensure that images across 99 percent of thefield-of-view are acquired within ±150nm (or less) relative to the bestfocal plane for each channel, and that images across more the entirefield-of-view are acquired within ±200nm (or less) relative to the bestfocal plane for each imaging channel.

Illumination optical path design: Another factor for improvingsignal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and/orincreasing throughput is to increase illumination power density to thesample. In some instances, the disclosed imaging systems may comprise anillumination path design that utilizes a high-power laser or laser diodecoupled with a liquid light guide. The liquid light guide removesoptical speckle that is intrinsic to coherent light sources such aslasers and laser diodes. Furthermore, the coupling optics are designedin such a way as to underfill the entrance aperture of the liquid lightguide. The underfilling of the liquid light guide entrance aperturereduces the effective numerical aperture of the illumination beamentering the objective lens, and thus improves light delivery efficiencythrough the objective onto the sample plane. With this designinnovation, one can achieve illumination power densities up to 3× thatfor conventional designs over a large field-of-view (FOV).

By utilizing the angle-dependent discrimination of s- andp-polarization, in some instances, the illumination beam polarizationmay be orientated to reduce the amount of back-scattered andback-reflected illumination light that reaches the imaging sensors.

Structured illumination systems: In some instances, the disclosedimaging modules and systems may comprise a structured illuminationoptical design to increase the effective spatial resolution of theimaging system and thus enable the use of higher surface densities ofclonally-amplified target nucleic acid sequences (clusters) on flow cellsurfaces for improved sequencing throughput. Structured illuminationmicroscopy (SIM) utilizes spatially structured (i.e., periodic) patternsof light for illumination of the sample plane and relies on thegeneration of interference patterns known as Moire fringes. Severalimages are acquired under slightly different illumination conditions,e.g., by shifting and/or rotating the pattern of the structuredillumination, to create the Moire fringes. Mathematical deconvolution ofthe resulting interference signal allows reconstruction of asuper-resolution image having up to about a two-fold improvement inspatial resolution over that achieved using diffraction-limited imagingoptics [Lutz (2011), “Biological Imaging by Superresolution LightMicroscopy”, Comprehensive Biotechnology (Second Ed.), vol. 1, pages579-589, Elsevier; Feiner-Gracia, et al. (2018), “15—Advanced OpticalMicroscopy Techniques for the Investigation of Cell-NanoparticleInteractions”, Smart Nanoparticles for Biomedicine: Micro and NanoTechnologies, pages 219-236, Elsevier; Nylk, et al. (2019), “Light-SheetFluorescence Microscopy With Structured Light”, Neurophotonics andBiomedical Spectroscopy, pages 477-501, Elsevier]. An example ofstructured illumination microscopy imaging systems has recently beendescribed in Hong, U.S. Patent Application Publication No. 2020/0218052.

FIG. 41 provides a non-limiting schematic illustration of an imagingsystem 4100 comprising a branched structured illumination optical designas disclosed herein. The first branch (or arm) of the illuminationoptical path of system 4100 comprises, e.g., a light source (lightemitter) 4110A, an optical collimator 4120A to collimate light emittedby light source 4110A, a diffraction grating 4130A in a firstorientation with respect to the optical axis, a rotating window 4140A,and a lens 4150A. The second branch of the illumination optical path ofsystem 4100 comprises, e.g., a light source 4110B, an optical collimator4120B to collimate light emitted by light source 4110B, a diffractiongrating 4130B in a second orientation with respect to the optical axis,a rotating window 4140B, and a lens 4150B. The diffraction gratings4130A and 4130B enable projection of patterns of light fringes on thesample plane.

In some instances, the light sources 4110A and 4110B may be incoherentlight sources (e.g., comprising one or more light emitting diodes(LEDs)) or coherent light sources (e.g., comprising one or more lasersor laser diodes). In some instances, the light sources 4110A and 4110Bmay comprise an optical fiber coupled to, e.g., an LED, laser, or laserdiode that outputs a light beam that is then collimated by therespective collimator lenses 4120A and 4120B. In some instances, lightsources 4110A and 4110B may output light of the same wavelength. In someinstances, light sources 4110A and 4110B may output light of differentwavelengths. Either of light sources 4110A and 4110B may be configuredto output light of any wavelength and/or wavelength range describedelsewhere herein. During imaging, light sources 4110A and 4110B may beswitched on or off using, for example, a high-speed shutter (not shown)positioned in the optical path or by pulsing the light sources at apredetermined frequency.

In the example shown in FIG. 41, the first illumination arm of system4100 includes a fixed vertical grating 4130A used to project a gratingpattern (e.g., a vertical light fringe pattern) in a first orientationonto the sample plane, e.g., a first interior surface 4188 of a flowcell 4187, and the second illumination arm includes a fixed horizontalgrating 4130B to project a grating pattern (e.g., a horizontal lightfringe pattern) in a second orientation onto the sample plane 4188.Advantageously, the diffraction gratings of imaging system 4100 do notneed to be mechanically rotated or translated during imaging in thisnon-limiting example, which may provide improved imaging speed, systemreliability, and system repeatability. In some instances, diffractiongratings 4130A and/or 4130B may be rotatable about their respectiveoptical axes such that the angle between the light fringe patternsprojected on the sample plane is adjustable.

As illustrated in FIG. 41, in some instances, diffraction gratings 4130Aand 4130B may be transmissive diffraction gratings that comprise aplurality of diffracting elements (e.g., parallel slits or grooves)formed in a glass substrate or other suitable surface. In someinstances, the gratings may be implemented as phase gratings thatprovide a periodic variation of the refractive index of the gratingmaterial. In some instances, the groove or feature spacing may be chosento diffract light at suitable angles and/or be tuned to the minimumresolvable feature size of the imaged samples for operation of imagingsystem 4100. In other instances, the diffraction gratings may bereflective diffraction gratings.

In the example illustrated in FIG. 41, the orientations of the verticaland horizontal light fringe patterns are offset by about 90 degrees. Inother instances, other orientations of the diffraction gratings may beused to create an offset of about 90 degrees. For example, thediffraction gratings may be oriented such that they project light fringepatterns that are offset ±45 degrees from the x or y axes of sampleplane (e.g., first interior flow cell surface) 4188. The configurationof imaging system 4100 illustrated in FIG. 41 may be particularlyadvantageous in the case of a sample support surface (e.g., an interiorsurface 4188 of a flow cell 4187) comprising regularly patternedfeatures laid out on a rectangular grid, as enhancement of imageresolution using the structured illumination approach can be achievedusing only two perpendicular grating orientations (e.g., the verticalgrating orientation and horizontal grating orientation).

Diffraction gratings 4130A and 4130B, in the example of system 4100, maybe configured to diffract the input illumination light beams into aseries of intensity maxima due to constructive interference according tothe relationship:

m=order number=d sin(θ)/λ

where d=the distance between slits or grooves in the diffractiongrating, θ=the angle of incidence of the illumination light relative toa normal to the surface of the diffraction grating, λ=the wavelength ofthe illumination light, and m=an integer value corresponding to anintensity maxima of the diffracted light, e.g., m=0, ±1, ±2, etc. Insome instances, a specific order of the diffracted illumination light,e.g., the first order (m=±1) light may be projected on the sample plane,e.g., interior flow cell surface 4188. In some instances, for example,vertical grating 4130A may diffract a collimated light beam into firstorder diffracted beams (±1 orders) which are focused onto the sampleplane in a first orientation, and horizontal grating 4130B may diffracta collimated light beam into first order diffracted beams which arefocused onto the sample plane in a second orientation. In someinstances, the zeroth order beam and/or all other higher order beams(e.g., m=±2 or higher) may be blocked, i.e., filtered out of theillumination pattern projected onto the sample plane 4188, using, forexample, a beam blocking element (not shown) such as an order filterthat may be inserted into the optical paths following the diffractiongratings.

Each branch of the structured illumination system in the example of 4100includes an optical phase modulator or phase shifter 4140A and 4140B tophase shift the diffracted light transmitted or reflected by each of thediffraction gratings 4130A and 4130B. During structured imaging, theoptical phase of each diffracted beam may be shifted by some fraction(e.g., ½, ½, ¼, etc.) of the pitch (X) of each fringe of the structuredpattern. In the example of FIG. 41, phase modulators 4140A and 4140B maybe implemented, e.g., as rotating optical phase plates actuated byrotatory actuators or other actuator mechanisms to rotate and modulatethe optical path-length of each diffracted beam. For example, opticalphase plate 4140A may be rotated about the vertical axis to shift theimage projected by vertical grating 4130A on sample plane 4188 left orright, and optical phase plate 4140B may rotate about the horizontalaxis to shift the image projected by horizontal grating 4130B on sampleplane 4188 in the perpendicular direction.

In other implementations, other types of phase modulators that changethe optical path length of the diffracted light (e.g., optical wedgesmounted on linear translation stages, etc.) may be used. Additionally,although optical phase modulators 4140A and 4140B are illustrated asbeing placed after diffraction gratings 4130A and 4130B, in otherimplementations they may be placed at other positions in theillumination optical path. In some instances, a single optical phasemodulator may be operated in two different directions to producedifferent light fringe patterns, or the position of a single opticalphase modulator may be adjusted using a single motion to simultaneouslyadjust the path lengths of both arms of the illumination optical path.

In the example illustrated in FIG. 41, optical component 4160 may beused to combine light from the two illumination optical paths. Opticalcomponent 4160 may comprise, for example, a partially-silvered mirror, adichroic mirror (depending on the wavelengths of light output by lightsources 4110A and 4110B), a mirror comprising a pattern of holes or apatterned reflective coating such that light from the two arms of theillumination system are combined in a lossless or nearly lossless manner(e.g., without significant loss of optical power other than a smallamount of absorption by the reflective coating), a polarizing beamsplitter (in the case that light sources 4110A and 4110B are configuredto produce polarized light), and the like. Optical component 4160 may belocated such that the desired diffracted orders of light reflected ortransmitted by each of the diffraction gratings are spatially resolved,and the unwanted orders of light are blocked. In some instances, opticalcomponent 4160 may pass the first order light output by the firstillumination light path and reflect the first order light output by thesecond illumination light path. In some instances, the structuredillumination pattern on the sample surface 4188 may be switched from avertical orientation (e.g., using diffraction grating 4130A) to ahorizontal orientation (e.g., using diffraction grating 4130B) byturning each light source on or off, or by opening and closing anoptical shutter in the optical path for the light source. In otherinstances, the structured illumination pattern may be switched by usingan optical switch to change the illumination optical path used toilluminate the sample plane.

Referring again to FIG. 41, a lens 4170, a semi-reflective mirror ordichroic mirror 4180, and an objective 4185 may be used to focus thestructured illumination light onto sample surface 4188 (e.g., the firstinterior surface of a flow cell 4187). Light that is emitted by,reflected by, or scattered by the sample surface 4188 is then collectedby objective 4185, transmitted through mirror 4180, and imaged by imagesensor or camera 4195. As noted, mirror 4180 may be a dichroic mirror toreflect structured illumination light received from each branch of theillumination optical path into objective 4185 for projection onto sampleplane 4188, and to pass through light emitted by the sample plane 4188(e.g., fluorescent light, which is emitted at different wavelengths thanthe excitation light) for imaging onto image sensor 4195.

In some instances, system 4100 may optionally comprise a custom tubelens 4190 as described elsewhere herein such that the focus of theimaging system may be shifted from the first interior surface 4188 tothe second interior surface 4189 of the flow cell 4187 to enable dualsurface imaging with minimal adjustment. In some instances, lens 4170may comprise a custom tube lens as described elsewhere herein such thatthe focus of the illumination optical path may be shifted from the firstinterior surface 4188 to the second interior surface 4189 of the flowcell 4187 to enable dual surface imaging with minimal adjustment. Insome instances, lens 4170 may be implemented to articulate along theoptical axis to adjust the focus of the structured illumination patternon the sample plane. In some instances, system 4100 may comprise anautofocus mechanism (not shown) to adjust focus of the illuminationlight and/or the focus of the image at the plane of image sensor 4195.In some instances, the system 4100 illustrated in FIG. 41 may provide ahigh optical efficiency due to the absence of a polarizer in the opticalpath. The use of unpolarized light may or may not have a significantimpact on illumination pattern contrast depending on the numericalaperture of objective 4185.

For the sake of simplicity, some optical components of imaging system4100 may have been omitted from FIG. 41 and the foregoing discussion.Although system 4100 is illustrated in this non-limiting example as asingle channel detection system, in other instances, it may beimplemented as a multi-channel detection system (e.g., using twodifferent image sensors and appropriate optics as well as light sourcesthat emit at two different wavelengths). Furthermore, although theillumination optical path of system 4100 is illustrated in thisnon-limiting example as comprising two branches, in some instances itmay be implemented as comprising, e.g., three branches, four branches,or more than four branches, each of which comprises a diffractiongrating at a fixed or adjustable relative orientation to each other.

In some instances, alternative illumination path optical designs may beused to create structured illumination. For example, in some instances,a single large, rotating optical phase modulator may be positioned afteroptical component 4160 and used in place of optical phase modulators4140A and 4140B to modulate the phases of both diffracted beams outputby the vertical and horizontal diffraction gratings 4130A and 4130B. Insome instances, instead of being parallel with respect to the opticalaxis of one of the diffraction gratings, the axis of rotation for thesingle rotating optical compensator may be offset by 45 degrees (oranother angular offset) from the optical axis of each of the verticaland horizontal diffraction gratings to allow for phase shifting alongboth illumination directions. In some instances, the single rotatingoptical phase modulator may be replaced by, e.g., a wedged opticalcomponent rotating about the nominal beam axis.

In another alternative illumination optical path design, diffractiongratings 4130A and 4130B may be mounted on respective linear motionstages so that they may be translated to change the optical path length(and thus the phase) of light reflected or transmitted by diffractiongratings 4130A and 4130B. The axis of motion of the linear motion stagesmay be perpendicular or otherwise offset from the orientation of theirrespective diffraction grating to provide translation of the diffractiongrating's fringe pattern along sample plane 4188. Suitable translationstages may comprise, e.g., crossed roller bearing stages, a linearmotor, a high-accuracy linear encoder, and/or other linear actuatortechnologies to provide precise linear translation of the diffractiongratings.

FIG. 42 provides a non-limiting example of a workflow for acquiring andprocessing imaged using structured illumination to enhance the spatialresolution of the imaging system. In some instances, the workflowillustrated in FIG. 42 may be performed to image an entire sample plane(e.g., an interior surface of a flow cell by means of image tiling) orto image a single area of a larger sample plane. The vertical 4130A andhorizontal 4130B diffraction gratings of the system 4100 illustrated inFIG. 41 may be used to project illumination light fringe patterns ontothe sample plane that have different known orientations and/or differentknown phase shifts. For example, the imaging system 4100 may usevertical grating 4130A and horizontal grating 4130B to generate thehorizontal and vertical illumination patterns respectively, whileoptical phase modulators 4140A and 4140B may be set to three differentpositions to produce the three phase shifts shown for each orientation.

During operation, a first illumination condition (e.g., a specificorientation of the diffraction grating and phase shift setting) may beused to project a grating light fringe pattern on the sample plane,e.g., flow cell surface. Following capture of an image using the firstillumination condition, one or more additional images acquired using oneor more phase shifted illumination patterns (e.g., 1, 2, 3, 4, 5, 6, ormore than 6 additional images acquired using 1, 2, 3, 4, 5, 6, or morethan 6 phase shifted illumination patterns) may be acquired. If theimaging system comprises a second branch of the illumination opticalpath, the image acquisition process may be repeated using a secondillumination condition as a starting point (e.g., a second specificorientation of the diffraction grating and phase shift setting), and theimage acquisition process may be repeated. In some instances, images maybe acquired for at least three different orientations of the diffractiongrating (e.g., spaced apart by 60 degrees relative to each other) usingat least 5 different phase shifted light fringe patterns. If no moreimages are to be acquired using different orientations of thediffraction grating or phase shifted illumination light fringe patterns,an image reconstruction algorithm may be used to process the acquiredimages and produce a reconstructed super-resolution image. In someinstances, images may be acquired for at least 1, 2, 3, 4, 5, 6, or morethan 6 different orientations of the diffraction grating using at least1, 2, 3, 4, 5, 6, or more than 6 different phase-shifted light fringepatterns at each orientation.

A potential disadvantage of acquiring multiple images for use inreconstructing single, super-resolution images is the time required toadjust the orientation and/or relative phase shift of the projectedlight fringe patterns and the exposure time required for acquiring eachimage, as well as the downstream image processing. Therefore, opticaldesigns that minimize the time required to change diffraction gratingorientation and relative phase, along with highly efficient imagereconstruction algorithms, are to be preferred. In some instances, fewerimages may be required to reconstruct super-resolution images of, e.g.,flow cell surfaces comprising discrete, fluorescently labeled clustersof amplified target nucleic acid sequences tethered to thelow-nonspecific binding surfaces described elsewhere herein than wouldordinarily be required for reconstructing higher resolution images ofconventional samples, e.g., stained tissue samples.

Referring again to FIG. 42, the afore-mentioned cycle may be repeatedfor different areas of a given flow cell surface, e.g., in the case thatthe images will be tiled to create a higher resolution image of theentire flow cell surface. In some instances, the afore-mentioned cyclemay be repeated after adjusting the focus of the imaging system if,e.g., a second flow cell surface is to be imaged.

Other super-resolution imaging techniques: In some instances, thedisclosed imaging systems may comprise the use of an alternativesuper-resolution imaging technique, e.g., photoactivation localizationmicroscopy (PALM), fluorescence photoactivation localization microscopy(FPALM), and/or stochastic optical reconstruction microscopy (STORM)see, for example, Lutz, et al. (2011), “Biological Imaging bySuperresolution Light Microscopy”, Comprehensive Biotechnology (SecondEd.), vol. 1, pages 579-589, Elsevier), which are based on statisticalcurve fitting of the intensity distribution observed in images of asingle molecule's point spread function (PSF) to a Gaussian distributionfunction. The Gaussian distribution function is then used to definelocation of the molecule in the sample plane with much higher precisionthan allowed by the classical resolution limit. The same approach may beused to image, e.g., small dispersed subsets of fluorescently labeledmolecules such as clonally amplified clusters of target nucleic acidsequences tethered to a low non-specific binding surface on a samplesupport or the interior surface of a flow cell.

The spatial accuracy or resolution achieved using these methods dependsupon the number of photons collected from the molecule before it isphotobleached and upon the background noise level [Lutz, et al. (2011),ibid]. In the case that background noise is negligible and collection ofat least 10,000 photons per molecule is possible, position accuracies of1-2 nm have been demonstrated. In some instances, e.g., using thesequencing-by-avidity approach described elsewhere herein,polymer-nucleotide conjugates comprising a plurality of fluorescentlabels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 labels perconjugate) to ensure a high photon count, optionally used in combinationwith the low non-specific binding surfaces disclosed elsewhere herein toensure very low background signals, may facilitate the use of thesesuper-resolution imaging techniques for genetic testing and sequencingapplications. Spatial accuracy or resolution decreases with decreasingnumbers of photons collected, however, even in the case that onlymoderate numbers of photons are collected, position location accuracy orresolution of 20 nm is possible. In some cases, an improvement of10-fold or better in lateral spatial resolution may be achieved. In somecases, an image resolution of better than 500 nm, 400 nm, 300 nm, 200nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or 10 nm may beachieved.

The second principle fundamental to this class of imaging is that smallnumbers of spatially separated fluorescent molecules within the sampleare imaged at any given time.

In some instances, the ability to control fluorescence emission ofsmall, dispersed subsets of fluorescent molecules in the sample plane iskey to facilitating super-resolution imaging. In the case offluorescence photoactivation localization microscopy (FPALM) andphotoactivation localization microscopy (PALM), for example, the use ofphotoactivatable green fluorescent proteins (PA-GFP) as a label hasallowed for controlled induction of fluorescent subsets in a sampleusing short pulses of 405 nm light to photoconvert the PA-GFP from adark, nonfluorescent state to a 488 nm excitable fluorescent state,thereby resulting in spatially separated subsets of fluorescentmolecules that can be imaged [Lutz, et al. (2011), ibid]. In the case ofstochastic optical reconstruction microscopy (STORM), thephoto-switching properties of, for example, the cyanine dye pairsCy5-Cy3 may be used in a similar fashion to enable the stochasticinduction of Cy5 fluorescence from a small subset of the molecules inthe sample at any given time, e.g., small subsets of molecules that arespatially separated by at least several resolution units. In someinstances, e.g., when combined with the sequencing-by-avidity approachdescribed elsewhere herein, polymer-nucleotide conjugates may comprise aphotoactivatable green fluorescent protein (PA-GFP) or a subdomain orportion thereof. In some instances, the polymer-nucleotide conjugatesmay comprise a mixture of conjugates in which a first portion is labeledwith, e.g., Cy3 labels, and a second portion is labeled with, e.g., Cy5labels. In some instances, the polymer nucleotide conjugates maycomprise a mixture of, e.g., Cy3 and Cy5 labels within the sameconjugate.

The super-resolved image is reconstructed from the sum of the Gaussianfits from all molecules or features (e.g., labeled nucleic acidclusters) imaged in a time stack of acquired images [Lutz, et al.(2011), ibid], where the intensity corresponds to the positionaluncertainty of the location of each molecule or subset of molecules.Unique to this kind of data set is the ability to render the image withdifferent localization precisions or resolutions. In some instances, animaging module comprising a total internal reflectance fluorescence(TIRF) optical imaging design may be advantageous in implementing theuse of these super-resolution imaging techniques as the evanescent waveused for excitation of fluorescence is restricted in the axial dimensionto less than 200 nm from the sample support or flow cell surface andthus suppresses background fluorescence signal. In some instances, theimaging system may comprise a higher numerical aperture objective thanutilized in other imaging module designs disclosed herein. The use ofhigher numerical aperture objectives may facilitate implementation ofevanescent wave excitation and highly efficient capture of photons fromthe fluorescent probes. In some instances, wide-field imaging usingsingle-photon-sensitive EM-CCD cameras or other types of image sensorsmay enable simultaneous imaging of many molecules or subsets ofmolecules (e.g., nucleic acid sequence clusters) per frame, therebyimproving the throughput of image acquisition.

In some instances, the data acquisition time required to acquire enoughimages for adequate feature definition and resolution may be shortenedby improvements in the sensitivity and speed of the imaging system,through the use of the sequencing-by-avidity reagents and lownon-specific binding surfaced disclosed herein to increase signal whilereducing or eliminating background, and the use of improved imagereconstruction algorithms.

Assessing image quality: For any of the embodiments of the opticalimaging designs disclosed herein, imaging performance or imaging qualitymay be assessed using any of a variety of performance metrics known tothose of skill in the art. Examples include, but are not limited to,measurements of modulation transfer function (MTF) at one or morespecified spatial frequencies, defocus, spherical aberration, chromaticaberration, coma, astigmatism, field curvature, image distortion,contrast-to-noise ratio (CNR), or any combination thereof

In some instances, the disclosed optical designs for dual-side imaging(e.g., the disclosed objective lens designs, tube lens designs, the useof an electro-optical phase plate in combination with an objective,etc., alone or in combination) may yield significant improvements forimage quality for both the upper (near) and lower (far) interiorsurfaces of a flow cell, such that the difference in an imagingperformance metric for imaging the upper interior surface and the lowerinterior surface of the flow cell is less than 20%, less than 15%, lessthan 10%, less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% for any of the imaging performance metrics listed above,either individually or in combination.

In some instances, the disclosed optical designs for dual-side imaging(e.g., comprising the disclosed tube lens designs, the use of anelectro-optical phase plate in combination with an objective, etc.) mayyield significant improvements for image quality such that an imagequality performance metric for dual-side imaging provides for an atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, or at least 30%improvement in the imaging performance metric for dual-side imagingcompared to that for a conventional system comprising, e.g., anobjective lens, a motion-actuated compensator (that is moved out of orinto the optical path when imaging the near or far interior surfaces ofa flow cell), and an image sensor for any of the imaging performancemetrics listed above, either individually or in combination. In someinstances, fluorescence imaging systems comprising one or more of thedisclosed tube lens designs provides for an at least equivalent orbetter improvement in an imaging performance metric for dual-sideimaging compared to that for a conventional system comprising anobjective lens, a motion-actuated compensator, and an image sensor. Insome instances, fluorescence imaging systems comprising one or more ofthe disclosed tube lens designs provides for an at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% improvement in an imagingperformance metric for dual-side imaging compared to that for aconventional system comprising an objective lens, a motion-actuatedcompensator, and an image sensor.

Imaging module specifications:

Excitation light wavelength(s): In any of the disclosed optical imagingmodule designs, the light source(s) of the disclosed imaging modules mayproduce visible light, such as green light and/or red light. In someinstances, the light source(s), alone or in combination with one or moreoptical components, e.g., excitation optical filters and/or dichroicbeam splitters, may produce excitation light at about 350 nm, 375 nm,400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm,625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm,850 nm, 875 nm, or 900 nm. Those of skill in the art will recognize thatthe excitation wavelength may have any value within this range, e.g.,about 620 nm.

Excitation light bandwidths: In any of the disclosed optical imagingmodule designs, the light source(s), alone or in combination with one ormore optical components, e.g., excitation optical filters and/ordichroic beam splitters, may produce light at the specified excitationwavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm,±80 nm, or greater. Those of skill in the art will recognize that theexcitation bandwidths may have any value within this range, e.g., about±18 nm.

Light source power output: In any of the disclosed optical imagingmodule designs, the output of the light source(s) and/or an excitationlight beam derived therefrom (including a composite excitation lightbeam) may range in power from about 0.5 W to about 5.0 W, or more (aswill be discussed in more detail below). In some instances, the outputof the light source and/or the power of an excitation light beam derivedtherefrom may be at least 0.5 W, at least 0.6 W, at least 0.7 W, atleast 0.8 W, at least 1 W, at least 1.1 W, at least 1.2 W, at least 1.3W, at least 1.4 W, at least 1.5 W, at least 1.6 W, at least 1.8 W, atleast 2.0 W, at least 2.2 W, at least 2.4 W, at least 2.6 W, at least2.8 W, at least 3.0 W, at least 3.5 W, at least 4.0 W, at least 4.5 W,or at least 5.0 W. In some implementations, the output of the lightsource and/or the power of an excitation light beam derived therefrom(including a composite excitation light beam) may be at most 5.0 W, atmost 4.5 W, at most 4.0 W, at most 3.5 W, at most 3.0 W, at most 2.8 W,at most 2.6 W, at most 2.4 W, at most 2.2 W, at most 2.0W, at most 1.8W, at most 1.6 W, at most 1.5 W, at most 1.4 W, at most 1.3 W, at most1.2 W, at most 1.1 W, at most 1 W, at most 0.8 W, at most 0.7 W, at most0.6 W, or at most 0.5 W. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the output of thelight source and/or the power of an excitation light beam derivedtherefrom (including a composite excitation light beam) may range fromabout 0.8 W to about 2.4 W. Those of skill in the art will recognizethat the output of the light source and/or the power of an excitationlight beam derived therefrom (including a composite excitation lightbeam) may have any value within this range, e.g., about 1.28 W.

Light source output power and CNR: In some implementations of thedisclosed optical imaging module designs, the output power of the lightsource(s) and/or the power of excitation light beam(s) derived therefrom(including a composite excitation light beam) is sufficient, incombination with an appropriate sample, to provide for acontrast-to-noise ratio (CNR) in images acquired by the illumination andimaging module of at least 5, at least 10, at least 15, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least30, at least 35, at least 40, or at least 50 or more, or any CNR withinany range formed by any of these values.

Fluorescence emission bands: In some instances, the disclosedfluorescence optical imaging modules may be configured to detectfluorescence emission produced by any of a variety of fluorophores knownto those of skill in the art. Examples of suitable fluorescence dyes foruse in, e.g., genotyping and nucleic acid sequencing applications (e.g.,by conjugation to nucleotides, oligonucleotides, or proteins) include,but are not limited to, fluorescein, rhodamine, coumarin, cyanine, andderivatives thereof, including the cyanine derivatives cyanine dye-3(Cy3), cyanine dye-5 (Cy5), cyanine dye-7 (Cy7), etc.

Fluorescence emission wavelengths: In any of the disclosed opticalimaging module designs, the detection channel or imaging channel of thedisclosed optical systems may include one or more optical components,e.g., emission optical filters and/or dichroic beam splitters,configured to collect emission light at about 350 nm, 375 nm, 400 nm,425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm,650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm,875 nm, or 900 nm. Those of skill in the art will recognize that theemission wavelength may have any value within this range, e.g., about825 nm.

Fluorescence emission light bandwidths: In any of the disclosed opticalimaging module designs, the detection channel or imaging channel maycomprise one or more optical components, e.g., emission optical filtersand/or dichroic beam splitters, configured to collect light at thespecified emission wavelength within a bandwidth of ±2 nm, ±5 nm, ±10nm, ±20 nm, ±40 nm, ±80 nm, or greater. Those of skill in the art willrecognize that the excitation bandwidths may have any value within thisrange, e.g., about ±18 nm.

Numerical aperture: In some instances, the numerical aperture of theobjective lens and/or optical imaging module (e.g., comprising anobjective lens and/or tube lens) in any of the disclosed optical systemdesigns may range from about 0.1 to about 1.4. In some instances, thenumerical aperture may be at least 0.1, at least 0.2, at least 0.3, atleast 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, atleast 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or atleast 1.4. In some instances, the numerical aperture may be at most 1.4,at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, atmost 0.2, or at most 0.1. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the numericalaperture may range from about 0.1 to about 0.6. Those of skill in theart will recognize that the numerical aperture may have any value withinthis range, e.g., about 0.55.

Optical resolution: In some instances, depending on the numericalaperture of the objective lens and/or optical system (e.g., comprisingan objective lens and/or tube lens), the minimum resolvable spot (orfeature) separation distance at the sample plane achieved by any of thedisclosed optical system designs may range from about 0.5 μm to about 2μm. In some instances, the minimum resolvable spot separation distanceat the sample plane may be at least 0.5 μm, at least 0.6 μm, at least0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 1.0μm. In some instances, the minimum resolvable spot separation distancemay be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm,at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe minimum resolvable spot separation distance may range from about 0.8μm to about 1.6 μm. Those of skill in the art will recognize that theminimum resolvable spot separation distance may have any value withinthis range, e.g., about 0.95 μm.

Optical resolution of first and second surfaces at different depths: Insome instances, the use of the novel objective lens and/or tube lensdesigns disclosed herein, in any of the optical modules or systemsdisclosed herein, may confer comparable optical resolution for first andsecond surfaces (e.g. the upper and lower interior surfaces of a flowcell) with or without the need to refocus between acquiring the imagesof the first and second surfaces. In some instances, the opticalresolution of the images thus obtained of the first and second surfacesmay be with 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of eachother, or within any value within this range.

Magnification: In some instances, the magnification of the objectivelens and/or tube lens, and/or optical system (e.g., comprising anobjective lens and/or tube lens) in any of the disclosed opticalconfigurations may range from about 2x to about 20x. In some instances,the optical system magnification may be at least 2×, at least 3×, atleast 4×, at least 5×, at least 6×, at least 7×, at least 8×, at least9×, at least 10×, at least 15×, or at least 20x. In some instances, theoptical system magnification may be at most 20×, at most 15×, at most10×, at most 9×, at most 8×, at most 7×, at most 6×, at most 5×, at most4×, at most 3×, or at most 2x. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances theoptical system magnification may range from about 3× to about 10×. Thoseof skill in the art will recognize that the optical system magnificationmay have any value within this range, e.g., about 7.5×.

Objective lens focal length: In some implementations of the disclosedoptical designs, the focal length of the objective lens may rangebetween 20 mm and 40 mm. In some instances, the focal length of theobjective lens may be at least 20 mm, at least 25 mm, at least 30 mm, atleast 35 mm, or at least 40 mm. In some instances, the focal length ofthe objective lens may be at most 40 mm, at most 35 mm, at most 30 mm,at most 25 mm, or at most 20 mm. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the focallength of the objective lens may range from 25 mm to 35 mm. Those ofskill in the art will recognize that the focal length of the objectivelens may have any value within the range of values specified above,e.g., about 37 mm.

Objective lens working distance: In some implementations of thedisclosed optical designs, the working distance of the objective lensmay range between about 100 μm and 30 mm. In some instances, the workingdistance may be at least 100 μm, at least 200 μm, at least 300 μm, atleast 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, atleast 800 μm, at least 900 μm, at least 1 mm, at least 2 mm, at least 4mm, at least 6 mm, at least 8 mm, at least 10 mm, at least 15 mm, atleast 20 mm, at least 25 mm, or at least 30 mm. In some instances, theworking distance may be at most 30 mm, at most 25 mm, at most 20 mm, atmost 15 mm, at most 10 mm, at most 8 mm, at most 6 mm, at most 4 mm, atmost 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm,at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most200 μm, at most 100 μm. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the working distanceof the objective lens may range from 500 μm to 2 mm. Those of skill inthe art will recognize that the working distance of the objective lensmay have any value within the range of values specified above, e.g.,about 1.25 mm.

Objectives optimized for imaging through thick coverslips: In someinstances of the disclosed optical designs, the design of the objectivelens may be improved or optimized for a different coverslip of flow cellthickness. For example, in some instances the objective lens may bedesigned for optimal optical performance for a coverslip that is fromabout 200 μm to about 1,000 μm thick. In some instances, the objectivelens may be designed for optimal performance with a coverslip that is atleast 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, atleast 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or atleast 1,000 μm thick. In some instances, the objective lens may bedesigned for optimal performance with a coverslip that is at most 1,000μm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, atmost 500 μm, at most 400 μm, at most 300 μm, or at most 200 μm thick.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the objective lens may be designed foroptimal optical performance for a coverslip that may range from about300 μm to about 900 μm. Those of skill in the art will recognize thatthe objective lens may be designed for optimal optical performance for acoverslip that may have any value within this range, e.g., about 725 μm.

Depth of field and depth of focus: In some instances, the depth of fieldand/or depth of focus for any of the disclosed imaging module (e.g.,comprising an objective lens and/or tube lens) designs may range fromabout 10 μm to about 800 μm, or more. In some instances, the depth offield and/or depth of focus may be at least 10 μm, at least 20 μm, atleast 30 μm, at least 40 μm, at least 50 μm, at least 75 μm, at least100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200μm, at least 250 μm, at least 300 μm, at least 300 μm, at least 400 μm,at least 500 μm, at least 600 μm, at least 700 μm, or at least 800 μm,or more. In some instances, the depth of field and/or depth of focus beat most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most400 μm, at most 300 μm, at most 250 μm, at most 200 μm, at most 175 μm,at most 150 μm, at most 125 μm, at most 100 μm, at most 75 μm, at most50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, orless. Any of the lower and upper values described in this paragraph maybe combined to form a range included within the present disclosure, forexample, in some instances the depth of field and/or depth of focus mayrange from about 100 μm to about 175 μm. Those of skill in the art willrecognize that the depth of field and/or depth of focus may have anyvalue within the range of values specified above, e.g., about 132 μm.

Field of view (FOV): In some implementations, the FOV of any of thedisclosed imaging module designs (e.g., that provided by a combinationof objective lens and detection channel optics (such as a tube lens))may range, for example, between about 1 mm and 5 mm (e.g., in diameter,width, length, or longest dimension). In some instances, the FOV may beat least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, atleast 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, or atleast 5.0 mm (e.g., in diameter, width, length, or longest dimension).In some instances, the FOV may be at most 5.0 mm, at most 4.5 mm, atmost 4.0 mm, at most 3.5 mm, at most 3.0 mm, at most 2.5 mm, at most 2.0mm, at most 1.5 mm, or at most 1.0 mm (e.g., in diameter, width, length,or longest dimension). Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the FOV may rangefrom about 1.5 mm to about 3.5 mm (e.g., in diameter, width, length, orlongest dimension). Those of skill in the art will recognize that theFOV may have any value within the range of values specified above, e.g.,about 3.2 mm (e.g., in diameter, width, length, or longest dimension).

Field-of-view (FOV) area: In some instances of the disclosed opticalsystem designs, the area of the field-of-view may range from about 2 mm²to about 5 mm². In some instances, the field-of-view may be at least 2mm², at least 3 mm², at least 4 mm², or at least 5 mm² in area. In someinstances, the field-of-view may be at most 5 mm², at most 4 mm², atmost 3 mm², or at most 2 mm² in area. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thefield-of-view may range from about 3 mm² to about 4 mm² in area. Thoseof skill in the art will recognize that the area of the field-of-viewmay have any value within this range, e.g., 2.75 mm².

Optimization of objective lens and/or tube lens MTF: In some instances,the design of the objective lens and/or at least one tube lens in thedisclosed imaging modules and systems is configured to optimize themodulation transfer function in the mid to high spatial frequency range.For example, in some instances, the design of the objective lens and/orat least one tube lens in the disclosed imaging modules and systems isconfigured to optimize the modulation transfer function in the spatialfrequency range from 500 cycles per mm to 900 cycles per mm, from 700cycles per mm to 1100 cycles per mm, from 800 cycles per mm to 1200cycles per mm, or from 600 cycles per mm to 1000 cycles per mm in thesample plane.

Optical aberration and diffraction-limited imaging performance: In someimplementations of any of the optical imaging module designs disclosedherein, the objective lens and/or tube lens may be configured to providethe imaging module with a field-of-view as indicated above such that theFOV has less than 0.15 waves of aberration over at least 60%, 70%, 80%,90%, or 95% of the field. In some implementations, the objective lensand/or tube lens may be configured to provide the imaging module with afield-of-view as indicated above such that the FOV has less than 0.1waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of thefield. In some implementations, the objective lens and/or tube lens maybe configured to provide the imaging module with a field-of-view asindicated above such that the FOV has less than 0.075 waves ofaberration over at least 60%, 70%, 80%, 90%, or 95% of the field. Insome implementations, the objective lens and/or tube lens may beconfigured to provide the imaging module with a field-of-view asindicated above such that the FOV is diffraction-limited over at least60%, 70%, 80%, 90%, or 95% of the field.

Angle of incidence of light beams on dichroic reflectors, beam splitter,and beam combiners: In some instances of the disclosed optical designs,the angles of incidence for a light beam incident on a dichroicreflector, beam splitter, or beam combiner may range between about 20degrees and about 45 degrees. In some instances, the angles of incidencemay be at least 20 degrees, at least 25 degrees, at least 30 degrees, atleast 35 degrees, at least 40 degrees, or at least 45 degrees. In someinstances, the angles of incidence may be at most 45 degrees, at most 40degrees, at most 35 degrees, at most 30 degrees, at most 25 degrees, orat most 20 degrees. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the angles of incidence mayrange from about 25 degrees to about 40 degrees. Those of skill in theart will recognize that the angles of incidence may have any valuewithin the range of values specified above, e.g., about 43 degrees.

Image sensor (photodetector array) size: In some instances, thedisclosed optical systems may comprise image sensor(s) having an activearea with a diagonal ranging from about 10 mm to about 30 mm, or larger.In some instances, the image sensors may have an active area with adiagonal of at least 10 mm, at least 12 mm, at least 14 mm, at least 16mm, at least 18 mm, at least 20 mm, at least 22 mm, at least 24 mm, atleast 26 mm, at least 28 mm, or at least 30 mm. In some instances, theimage sensors may have an active area with a diagonal of at most 30 mm,at most 28 mm, at most 26 mm, at most 24 mm, at most 22 mm, at most 20mm, at most 18 mm, at most 16 mm, at most 14 mm, at most 12 mm, or atmost 10 mm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the image sensor(s) may havean active area with a diagonal ranging from about 12 mm to about 24 mm.Those of skill in the art will recognize that the image sensor(s) mayhave an active area with a diagonal having any value within the range ofvalues specified above, e.g., about 28.5 mm.

Image sensor pixel size and pitch: In some instances, the pixel sizeand/or pitch selected for the image sensor(s) used in the disclosedoptical system designs may range in at least one dimension from about 1μm to about 10 μm. In some instances, the pixel size and/or pitch may beat least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or atleast 10 μm. In some instances, the pixel size and/or pitch may be atmost 10 μm, at most 9 μm, at most 8 at most 7 μm, at most 6 μm, at most5 μm, at most 4 μm, at most 3 μm, at most 2 μm, or at most 1 μm. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the pixel size and/or pitch may range from about 3 p.m toabout 9 μm. Those of skill in the art will recognize that the pixel sizeand/or pitch may have any value within this range, e.g., about 1.4 μm.

Oversampling: In some instances of the disclosed optical designs, aspatial oversampling scheme is utilized wherein the spatial samplingfrequency is at least 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×,or 10x the optical resolution X (lp/mm).

Maximum translation stage velocity: In some instances of the disclosedoptical imaging modules, the maximum translation stage velocity on anyone axis may range from about 1 mm/sec to about 5 mm/sec. In someinstances, the maximum translation stage velocity may be at least 1mm/sec, at least 2 mm/sec, at least 3 mm/sec, at least 4 mm/sec, or atleast 5 mm/sec. In some instances, the maximum translation stagevelocity may be at most 5 mm/sec, at most 4 mm/sec, at most 3 mm/sec, atmost 2 mm/sec, or at most 1 mm/sec. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances themaximum translation stage velocity may range from about 2 mm/sec toabout 4 mm/sec. Those of skill in the art will recognize that themaximum translation stage velocity may have any value within this range,e.g., about 2.6 mm/sec.

Maximum translation stage acceleration: In some instances of thedisclosed optical imaging modules, the maximum acceleration on any oneaxis of motion may range from about 2 mm/sec² to about 10 mm/sec². Insome instances, the maximum acceleration may be at least 2 mm/sec², atleast 3 mm/sec², at least 4 mm/sec², at least 5 mm/sec², at least 6mm/sec², at least 7 mm/sec², at least 8 mm/sec², at least 9 mm/sec², orat least 10 mm/sec². In some instances, the maximum acceleration may beat most 10 mm/sec², at most 9 mm/sec², at most 8 mm/sec², at most 7mm/sec², at most 6 mm/sec², at most 5 mm/sec², at most 4 mm/sec², atmost 3 mm/sec², or at most 2 mm/sec². Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances themaximum acceleration may range from about 2 mm/sec² to about 8 mm/sec².Those of skill in the art will recognize that the maximum accelerationmay have any value within this range, e.g., about 3.7 mm/sec².

Translation stage positioning repeatability: In some instances of thedisclosed optical imaging modules, the repeatability of positioning forany one axis may range from about 0.1 μm to about 2 In some instances,the repeatability of positioning may be at least 0.1 μm, at least 0.2μm, at least 0.3 μm, at least 0.4 μm, at least 0.5 μm, at least 0.6 μm,at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, atleast 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or atleast 2.0 μm. In some instances, the repeatability of positioning may beat most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm,at most 0.6 μm, at most 0.5 μm, at most 0.4 μm, at most 0.3 μm, at most0.2 μm, or at most 0.1 μm. Any of the lower and upper values describedin this paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the repeatability ofpositioning may range from about 0.3 μm to about 1.2 μm. Those of skillin the art will recognize that the repeatability of positioning may haveany value within this range, e.g., about 0.47 μm.

FOV repositioning time: In some instances of the disclosed opticalimaging modules, the maximum time required to reposition the sampleplane (field-of-view) relative to the optics, or vice versa, may rangefrom about 0.1 sec to about 0.5 sec. In some instances, the maximumrepositioning time (i.e., the scan stage step and settle time) may be atleast 0.1 sec, at least 0.2 sec, at least 0.3 sec, at least 0.4 sec, orat least 0.5 sec. In some instances, the maximum repositioning time maybe at most 0.5 sec, at most 0.4 sec, at most 0.3 sec, at most 0.2 sec,or at most 0.1 sec. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the maximum repositioningtime may range from about 0.2 sec to about 0.4 sec. Those of skill inthe art will recognize that the maximum repositioning time may have anyvalue within this range, e.g., about 0.45 sec.

Error threshold for autofocus correction: In some instances of thedisclosed optical imaging modules, the specified error threshold fortriggering an autofocus correction may range from about 50 nm to about200 nm. In some instances, the error threshold may be at least 50 nm, atleast 75 nm, at least 100 nm, at least 125 nm, at least 150 nm, at least175 nm, or at least 200 nm. In some instances, the error threshold maybe at most 200 nm, at most 175 nm, at most 150 nm, at most 125 nm, atmost 100 nm, at most 75 nm, or at most 50 nm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe error threshold may range from about 75 nm to about 150 nm. Those ofskill in the art will recognize that the error threshold may have anyvalue within this range, e.g., about 105 nm.

Image acquisition time: In some instances of the disclosed opticalimaging modules, the image acquisition time may range from about 0.001sec to about 1 sec. In some instances, the image acquisition time may beat least 0.001 sec, at least 0.01 sec, at least 0.1 sec, or at least 1sec. in some instances, the image acquisition time may be at most 1 sec,at most 0.1 sec, at most 0.01 sec, or at most 0.001 sec. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the image acquisition time may range from about 0.01 secto about 0.1 sec. Those of skill in the art will recognize that theimage acquisition time may have any value within this range, e.g., about0.250 seconds.

Imaging time per FOV: In some instances, the imaging times may rangefrom about 0.5 seconds to about 3 seconds per field-of-view. In someinstances, the imaging time may be at least 0.5 seconds, at least 1second, at least 1.5 seconds, at least 2 seconds, at least 2.5 seconds,or at least 3 seconds per FOV. In some instances, the imaging time maybe at most 3 seconds, at most 2.5 seconds, at most 2 seconds, at most1.5 seconds, at most 1 second, or at most 0.5 seconds per FOV. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the imaging time may range from about 1 second to about2.5 seconds. Those of skill in the art will recognize that the imagingtime may have any value within this range, e.g., about 1.85 seconds.

Flatness offield: In some instances, images across 80%, 90%, 95%, 98%,99%, or 100% percent of the field-of-view are acquired within ±200 nm,±175 nm, ±150 nm, ±125 nm, 100 nm, ±75 nm, or ±50 nm relative to thebest focal plane for each fluorescence (or other imaging mode) detectionchannel.

Systems and system components for genomics and other applications: Asnoted above, in some implementations, the disclosed optical imagingmodules may function as modules, components, sub-assemblies, orsub-systems of larger systems configured for performing, e.g., genomicsapplications (e.g., genetic testing and/or nucleic acid sequencingapplications) or other chemical analysis, biochemical analysis, nucleicacid analysis, cell analysis or tissue analysis applications. FIG. 39provides a non-limiting example of a block diagram for, e.g., asequencing system as disclosed herein. In addition to one, two, three,four, or more than four imaging modules as disclosed herein (each ofwhich may comprise one or more illumination optical paths and/or one ormore detection optical paths (e.g., one or more detection channelsconfigured for imaging fluorescence emission within a specifiedwavelength range onto an image sensor)), such systems may comprise oneor more X-Y translation stages, one or more X-Y-Z translation stages,flow cells or cartridges, fluidics systems and fluid flow controlmodules, reagent cartridges, temperature control modules, fluiddispensing robotics, cartridge- and/or microplate-handling(pick-and-place) robotics, light-tight housings and/or environmentalcontrol chambers, one or more processors or computers, data storagemodules, data communication modules (e.g., Bluetooth, WiFi, intranet, orintern& communication hardware and associated software), displaymodules, one or more local and/or cloud-based software packages (e.g.,instrument/system control software packages, image processing softwarepackages, data analysis software packages), etc., or any combinationthereof

Translation stages: In some implementations of the imaging and analysissystems (e.g., nucleic acid sequencing systems) disclosed herein, thesystem may comprise one or more (e.g., one, two, three, four, or morethan four) high precision X-Y (or in some cases, X-Y-Z) translationstage(s) for re-positioning one or more sample support structure(s)(e.g., flow cell(s)) in relation to the one or more imaging modules, forexample, in order to tile one or more images, each corresponding to afield-of-view of the imaging module, to reconstruct composite image(s)of an entire flow cell surface. In some implementations of the imagingsystems and genomics analysis systems (e.g., nucleic acid sequencingsystems) disclosed herein, the system may comprise one or more (e.g.,one, two, three, four, or more than four) high precision X-Y (or in somecases, X-Y-Z) translation stage(s) for re-positioning the one or moreimaging modules in relation to one or more sample support structure(s)(e.g., flow cell(s)), for example, in order to tile one or more images,each corresponding to a field-of-view of the imaging module, toreconstruct composite image(s) of an entire flow cell surface.

Suitable translation stages are commercially available from a variety ofvendors, for example, Parker Hannifin. Precision translation stagesystems typically comprise a combination of several componentsincluding, but not limited to, linear actuators, optical encoders, servoand/or stepper motors, and motor controllers or drive units. Highprecision and repeatability of stage movement is required for thesystems and methods disclosed herein in order to ensure accurate andreproducible positioning and imaging of, e.g., fluorescence signals wheninterspersing repeated steps of reagent delivery and optical detection.

Consequently, the systems disclosed herein may comprise specifying theprecision with which the translation stage is configured to position asample support structure in relation to the illumination and/or imagingoptics (or vice versa). In one aspect of the present disclosure, theprecision of the one or more translation stages is between about 0.1 μmand about 10 μm. In other aspects, the precision of the translationstage is about 10 μm or less, about 9 μm or less, about 8 μm or less,about 7 μm or less, about 6 μm or less, about 5 μm or less, about 4 μmor less, about 3 μm or less, about 2 μm or less, about 1 μm or less,about 0.9 μm or less, about 0.8 μm or less, about 0.7 μm or less, about0.6 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μmor less, about 0.2 μm or less, or about 0.1 μm or less. Those of skillin the art will appreciate that, in some instances, the positioningprecision of the translation stage may fall within any range bounded byany of two of these values (e.g. from about 0.5 μm to about 1.5 μm). Insome instances, the positioning precision of the translation stage mayhave any value within the range of values included in this paragraph,e.g., about 0.12 μm.

Flow cells, microfluidic devices, and cartridges: The flow cell devicesand flow cell cartridges disclosed herein may be used as components ofsystems designed for a variety of chemical analysis, biochemicalanalysis, nucleic acid analysis, cell analysis, or tissue analysisapplication. In general, such systems may comprise one or more one ormore of the disclosed single capillary flow cell devices, multiplecapillary flow cell devices, capillary flow cell cartridges, and/ormicrofluidic devices and cartridges described herein. Additionaldescription of the disclosed flow cell devices and cartridges may befound in PCT Patent Application Publication WO 2020/118255, which isincorporated herein by reference in its entirety.

In some instances, the systems disclosed herein may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 single capillary flow cell devices,multiple capillary flow cell devices, capillary flow cell cartridges,and/or microfluidic devices and cartridges. In some instances, thesingle capillary flow cell devices, multiple capillary flow celldevices, and/or microfluidic devices and cartridges may be fixedcomponents of the disclosed systems. In some instances, the singlecapillary flow cell devices, multiple capillary flow cell devices,and/or microfluidic devices and cartridges may be removable,exchangeable components of the disclosed systems. In some instances, thesingle capillary flow cell devices, multiple capillary flow celldevices, and/or microfluidic devices and cartridges may be disposable orconsumable components of the disclosed systems.

In some implementations, the disclosed single capillary flow celldevices (or single capillary flow cell cartridges) comprise a singlecapillary, e.g., a glass or fused-silica capillary, the lumen of whichforms a fluid flow path through which reagents or solutions may flow,and the interior surface of which may form a sample support structure towhich samples of interest are bound or tethered. In someimplementations, the multi-capillary capillary flow cell devices (ormulti-capillary flow cell cartridges) disclosed herein may comprise 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morethan 20 capillaries configured for performing an analysis technique thatfurther comprises imaging as a detection method.

In some instances, one or more capillaries may be packaged within achassis to form a cartridge that facilitates ease-of-handling,incorporates adapters or connectors for making external fluidconnections, and may optionally include additional integratedfunctionality such as reagent reservoirs, waste reservoirs, valves(e.g., microvalves), pumps (e.g., micropumps), etc., or any combinationthereof.

FIG. 29 illustrates one non-limiting example of a single glass capillaryflow cell device that comprises two fluidic adaptors—one affixed to eachend of the piece of glass capillary—that are designed to mate withstandard OD fluidic tubing to provide for convenient, interchangeablefluid connections with an external fluid flow control system. Thefluidic adaptors can be attached to the capillary using any of a varietyof techniques known to those of skill in the art including, but notlimited to, press fit, adhesive bonding, solvent bonding, laser welding,etc., or any combination thereof

In general, the capillaries used in the disclosed capillary flow celldevices and capillary flow cell cartridges will have at least oneinternal, axially-aligned fluid flow channel (or “lumen”) that runs thefull length of the capillary. In some instances, the capillary may havetwo, three, four, five, or more than five internal, axially-alignedfluid flow channels (or “lumen”).

A number specified cross-sectional geometries for suitable capillaries(or the lumen thereof) are consistent with the disclosure hereinincluding, but not limited to, circular, elliptical, square,rectangular, triangular, rounded square, rounded rectangular, or roundedtriangular cross-sectional geometries. In some instances, the capillary(or lumen thereof) may have any specified cross-sectional dimension orset of dimensions. For example, in some instances the largestcross-sectional dimension of the capillary lumen (e.g. the diameter ifthe lumen is circular in shape, or the diagonal if the lumen is squareor rectangular in shape) may range from about 10 um to about 10 mm. Insome aspects, the largest cross-sectional dimension of the capillarylumen may be at least 10 um, at least 25 um, at least 50 um, at least 75um, at least 100 um, at least 200 um, at least 300 um, at least 400 um,at least 500 um, at least 600 um, at least 700 um, at least 800 um, atleast 900 um, at least 1 mm, at least 2 mm, at least 3 mm, at least 4mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least9 mm, or at least 10 mm. In some aspects, the largest cross-sectionaldimension of the capillary lumen may be at most 10 mm, at most 9 mm, atmost 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, atmost 3 mm, at most 2 mm, at most 1 mm, at most 900 um, at most 800 um,at most 700 um, at most 600 um, at most 500 um, at most 400 um, at most300 um, at most 200 um, at most 100 um, at most 75 um, at most 50 um, atmost 25 um, or at most 10 um. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thelargest cross-sectional dimension of the capillary lumen may range fromabout 100 um to about 500 um. Those of skill in the art will recognizethat the largest cross-sectional dimension of the capillary lumen mayhave any value within this range, e.g., about 124 um.

In some instances, e.g., wherein the lumen of the one or morecapillaries in a flow cell device or cartridge has a square orrectangular cross-section, the distance between a first interior surface(e.g., a top or upper surface) and a second interior surface (e.g., abottom or lower surface) that defines the gap height or thickness of afluid flow channel may range from about 10 p.m to about 500 μm. In someinstances, the gap height may be at least 10 μm, at least 20 μm, atleast 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, atleast 150 μm, at least 175 μm, at least 200 μm, at least 225 μm, atleast 250 μm, at least 275 μm, at least 300 μm, at least 325 μm, atleast 350 μm, at least 375 μm, at least 400 μm, at least 425 μm, atleast 450 μm, at least 475 μm, or at least 500 μm. In some instances,the gap height may be at most 500 μm, at most 475 μm, at most 450 p.m,at most 425 μm, at most 400 μm, at most 375 μm, at most 350 μm, at most325 μm, at most 300 μm, at most 275 μm, at most 250 μm, at most 225 μm,at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most100 μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, atmost 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, or most 10 μm.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the gap height may range from about 40 μm toabout 125 μm. Those of skill in the art will recognize that the gapheight may have any value within the range of values in this paragraph,e.g., about 122 μm.

In some instances, the length of the one or more capillaries used tofabricate the disclosed capillary flow cell devices or flow cellcartridges may range from about 5 mm to about 5 cm or greater. In someinstances, the length of the one or more capillaries may be less than 5mm, at least 5 mm, at least 1 cm, at least 1.5 cm, at least 2 cm, atleast 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least4.5 cm, or at least 5 cm. In some instances, the length of the one ormore capillaries may be at most 5 cm, at most 4.5 cm, at most 4 cm, atmost 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, at most 1.5 cm,at most 1 cm, or at most 5 mm. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the lengthof the one or more capillaries may range from about 1.5 cm to about 2.5cm. Those of skill in the art will recognize that the length of the oneor more capillaries may have any value within this range, e.g., about1.85 cm. In some instances, devices or cartridges may comprise aplurality of two or more capillaries that are the same length. In someinstances, devices or cartridges may comprise a plurality of two or morecapillaries that are of different lengths.

The capillaries used for constructing the disclosed capillary flow celldevices or capillary flow cell cartridges may be fabricated from any ofa variety of materials known to those of skill in the art including, butnot limited to, glass (e.g., borosilicate glass, soda lime glass, etc.),fused silica (quartz), polymer (e.g., polystyrene (PS), macroporouspolystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC),polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),cyclic olefin polymers (COP), cyclic olefin copolymers (COC),polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.),polyetherimide (PEI) and perfluoroelastomer (FFKM) as more chemicallyinert alternatives, or any combination thereof. PEI is somewhere betweenpolycarbonate and PEEK in terms of both cost and chemical compatibility.FFKM is also known as Kalrez.

The one or more materials used to fabricate the capillaries are oftenoptically transparent to facilitate use with spectroscopic orimaging-based detection techniques. In some instances, the entirecapillary will be optically transparent. Alternately, in some instances,only a portion of the capillary (e.g., an optically transparent“window”) will be optically transparent.

The capillaries used for constructing the disclosed capillary flow celldevices and capillary flow cell cartridges may be fabricated using anyof a variety of techniques known to those of skill in the art, where thechoice of fabrication technique is often dependent on the choice ofmaterial used, and vice versa. Examples of suitable capillaryfabrication techniques include, but are not limited to, extrusion,drawing, precision computer numerical control (CNC) machining andboring, laser photoablation, and the like.

In some implementations, the capillaries used in the disclosed capillaryflow cell devices and cartridges may be off-the-shelf commercialproducts. Examples of commercial vendors that provide precisioncapillary tubing include Accu-Glass (St. Louis, MO; precision glasscapillary tubing), Polymicro Technologies (Phoenix, AZ; precision glassand fused-silica capillary tubing), Friedrich & Dimmock, Inc.(Millville, NJ; custom precision glass capillary tubing), and DrummondScientific (Broomall, PA; OEM glass and plastic capillary tubing).

The fluidic adapters that are attached to the capillaries of thecapillary flow cell devices and cartridges disclosed herein, and othercomponents of the capillary flow cell devices or cartridges, may befabricated using any of a variety of suitable techniques (e.g.,extrusion molding, injection molding, compression molding, precision CNCmachining, etc.) and materials (e.g., glass, fused-silica, ceramic,metal, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET), etc.), where again the choice of fabricationtechnique is often dependent on the choice of material used, and viceversa.

FIG. 30 provides a non-limiting example of capillary flow cell cartridgethat comprises two glass capillaries, fluidic adaptors (two percapillary in this example), and a cartridge chassis that mates with thecapillaries and/or fluidic adapters such that the capillaries are heldin a fixed orientation relative to the cartridge. In some instances, thefluidic adaptors may be integrated with the cartridge chassis. In someinstances, the cartridge may comprise additional adapters that mate withthe capillaries and/or capillary fluidic adapters. As noted elsewhereherein, in some instances, the cartridge may comprise additionalfunctional components. In some instances, the capillaries arepermanently mounted in the cartridge. In some instances, the cartridgechassis is designed to allow one or more capillaries of the flow cellcartridge to be interchangeable removed and replaced. For example, insome instances, the cartridge chassis may comprise a hinged “clamshell”configuration which allows it to be opened so that one or morecapillaries may be removed and replaces. In some instances, thecartridge chassis is configured to mount on, for example, the stage of afluorescence microscope or within a cartridge holder of a fluorescenceimaging module or instrument system of the present disclosure.

In some instances, the disclosed flow cell devices may comprisemicrofluidic devices (or “microfluidic chips”) and cartridges, where themicrofluidic devices are fabricated by forming fluid channels in one ormore layers of a suitable material and comprise one or more fluidchannels (e.g., “analysis” channels) configured for performing ananalysis technique that further comprises imaging as a detection method.In some implementations, the microfluidic devices or cartridgesdisclosed herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more than 20 fluid channels (e.g.,“analysis” fluid channels) configured for performing an analysistechnique that further comprises imaging as a detection method. In someinstances, the disclosed microfluidic devices may further compriseadditional fluid channels (e.g., for dilution or mixing of reagents),reagent reservoirs, waste reservoirs, adapters for making external fluidconnections, and the like, to provide integrated “lab-on-a-chip”functionality within the device.

A non-limiting example of microfluidic flow cell cartridge comprises achip having two or more parallel glass channels formed on the chip,fluidic adaptors coupled to the chip, and a cartridge chassis that mateswith the chip and/or fluidic adapters such that the chip is posited in afixed orientation relative to the cartridge. In some instances, thefluidic adaptors may be integrated with the cartridge chassis. In someinstances, the cartridge may comprise additional adapters that mate withthe chip and/or fluidic adapters. In some instances, the chip ispermanently mounted in the cartridge. In some instances, the cartridgechassis is designed to allow one or more chips of the flow cellcartridge to be interchangeably removed and replaced. For example, insome instances, the cartridge chassis may comprise a hinged “clamshell”configuration which allows it to be opened so that one or more chips maybe removed and replaces. In some instances, the cartridge chassis isconfigured to mount on, for example, the stage of a microscope system orwithin a cartridge holder of an imaging system. Even through only onechip is described in the non-limiting example, it is understood thatmore than one chip can be used in the microfluidic flow cell cartridge.The flow cell cartridges of the present disclosure may comprise a singlemicrofluidic chip or a plurality of microfluidic chips. In someinstances, the flow cell cartridges of the present disclosure maycomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more than 20 microfluidic chips. The packaging of one or moremicrofluidic devices within a cartridge may facilitate ease-of-handlingand correct positioning of the device within the optical imaging system.

The fluid channels within the disclosed microfluidic devices andcartridges may have an of a variety of cross-sectional geometriesincluding, but not limited to, circular, elliptical, square,rectangular, triangular, rounded square, rounded rectangular, or roundedtriangular cross-sectional geometries. In some instances, the fluidchannels may have any specified cross-sectional dimension or set ofdimensions. For example, in some instances, the height (e.g., gapheight), width, or largest cross-sectional dimension of the fluidchannels (e.g., the diagonal if the fluid channel has a square, roundedsquare, rectangular, or rounded rectangular cross-section) may rangefrom about 10 μm to about 10 mm. In some aspects, the height (e.g., gapheight), width, or largest cross-sectional dimension of the fluidchannels may be at least 10 μm, at least 25 μm, at least 50 μm, at least75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm,at least 900 μm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least9 mm, or at least 10 mm. In some aspects, the height (e.g., gap height),width, or largest cross-sectional dimension of the fluid channels may beat most 10 mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm,at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, atmost 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, atmost 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the height (e.g., gap height), width, or largestcross-sectional dimension of the fluid channels may range from about 20μm to about 200 μm. Those of skill in the art will recognize that theheight (e.g., gap height), width, or largest cross-sectional dimensionof the fluid channels may have any value within this range, e.g., about122 μm.

In some instances, the length of the fluid channels in the disclosedmicrofluidic devices and cartridges may range from about 5 mm to about10 cm or greater. In some instances, the length of the fluid channelsmay be less than 5 mm, at least 5 mm, at least 1 cm, at least 1.5 cm, atleast 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4cm, at least 4.5 cm, at least 5 cm, at least 6 cm, at least 7 cm, atleast 8 cm, at least 9 cm, or at least 10 cm. In some instances, thelength of the fluid channels may be at most 10 cm, at most 9 cm, at most8 cm, at most 7 cm, at most 6 cm, at most 5 cm, at most 4.5 cm, at most4 cm, at most 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, atmost 1.5 cm, at most 1 cm, or at most 5 mm. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe length of the fluid channels may range from about 1.5 cm to about2.5 cm. Those of skill in the art will recognize that the length of thefluid channels may have any value within this range, e.g., about 1.35cm. In some instances, the microfluidic devices or cartridges maycomprise a plurality of fluid channels that are the same length. In someinstances, the microfluidic devices or cartridges may comprise aplurality of fluid channels that are of different lengths.

The disclosed microfluidic devices will comprise at least one layer ofmaterial having one or more fluid channels formed therein. In someinstances, the microfluidic chip may include two layers bonded togetherto form one or more fluid channels. In some instances, the microfluidicchip may include three or more layers bonded together to form one ormore fluid channels. In some instances, the microfluidic fluid channelsmay have an open top. In some instances, the microfluidic fluid channelsmay be fabricated within one layer, e.g., the top surface of a bottomlayer, and sealed by bonding the top surface of the bottom layer to thebottom surface of a top layer of material. In some instances, themicrofluidic channels may be fabricated within one layer, e.g., aspatterned channels the depth of which extends through the full thicknessof the layer, which is then sandwiched between and bonded to twonon-patterned layers to seal the fluid channels. In some instances, themicrofluidic channels are fabricated by the removal of a sacrificiallayer on the surface of a substrate. This method does not require thebulk substrate (e.g., a glass or silicon wafer) to be etched away.Instead, the fluid channels are located on the surface of the substrate.In some instances, the microfluidic channels may be fabricated in or onthe surface of a substrate and then sealed by deposition of a conformalfilm or layer on the surface of the substrate to create sub-surface orburied fluid channels in the chip.

The microfluidic chips can be manufactured using a combination ofmicrofabrication processes. Because the devices are microfabricated,substrate materials will typically be selected based upon theircompatibility with known microfabrication techniques, e.g.,photolithography, wet chemical etching, laser ablation, laserirradiation, air abrasion techniques, injection molding, embossing, andother techniques. The substrate materials are also generally selectedfor their compatibility with the full range of conditions to which themicrofluidic devices may be exposed, including extremes of pH,temperature, salt concentration, and application of electromagnetic(e.g. light) or electric fields.

The disclosed microfluidic chips may be fabricated from any of a varietyof materials known to those of skill in the art including, but notlimited to, glass (e.g., borosilicate glass, soda lime glass, etc.),fused-silica (quartz), silicon, a polymer (e.g., polystyrene (PS),macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),polycarbonate (PC), polypropylene (PP), polyethylene (PE), high densitypolyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefincopolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane(PDMS), etc.), polyetherimide (PEI) and perfluoroelastomer (FFKM) (asmore chemically inert alternatives), or any combination thereof. In somepreferred instances, the substrate material(s) may include silica-basedsubstrates, such as borosilicate glass, and quartz, as well as othersubstrate materials.

The disclosed microfluidic devices may be fabricated using any of avariety of techniques known to those of skill in the art, where thechoice of fabrication technique is often dependent on the choice ofmaterial used, and vice versa. The microfluidic channels on the chip canbe constructed using techniques suitable for forming micro-structures ormicro-patterns on the surface of a substrate. In some instances, thefluid channels are formed by laser irradiation. In some instances, themicrofluidic channels are formed by focused femtosecond laser radiation.In some instances, the microfluidic channels are formed byphotolithography and etching including, but not limited to, chemicaletching, plasma etching, or deep reactive ion etching. In someinstances, the microfluidic channels are formed using laser etching. Insome instances, the microfluidic channels are formed using adirect-write lithography technique. Examples of direct-write lithographyinclude electron beam direct-write and focused ion beam milling.

In additional preferred instances, the substrate material(s) maycomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, and the like. Such polymeric substrates may be readilypatterned or micromachined using available microfabrication techniques,such as those described above. In some instances, microfluidic chips maybe fabricated from polymeric materials, e.g., from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing, stamping, or by polymerizing the polymeric precursor materialwithin a mold (see, e.g., U.S. Pat. No. 5,512,131). In some instances,such polymeric substrate materials are preferred for their ease ofmanufacture, low cost, and disposability, as well as their generalinertness to most extreme reaction conditions. As with flow cell devicesfabricated from other materials, e.g., glass, flow cell devicesfabricated from these polymeric materials may include treated surfaces,e.g., derivatized or coated surfaces, to enhance their utility in themicrofluidic system, as will be discussed in more detail below.

The fluid channels and/or fluid chambers of the microfluidic devices aretypically fabricated into the upper surface of a first substrate asmicroscale channels (e.g., grooves, indentations, etc.) using the abovedescribed microfabrication techniques. The first substrate comprises atop side having a first planar surface and a bottom side. In themicrofluidic devices prepared in accordance with the methods describedherein, the plurality of fluid channels (e.g., grooves and/orindentations) are formed on the first planar surface. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surface (prior to bonding to a second substrate) have abottom and side walls, with the top remaining open. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surface (prior to bonding to a second substrate) have abottom and side walls and the top remaining closed. In some instances,the fluid channels (e.g., grooves and/or indentations) formed in thefirst planar surfaces (prior to bonding to a second substrate) have onlyside walls and no top or bottom surface (i.e., the fluid channels spanthe full thickness of the first substrate.

Fluid channels and chambers may be sealed by placing the first planarsurface of the first substrate in contact with, and bonding to, theplanar surface of a second substrate to form the channels and/orchambers (e.g., the interior portion) of the device at the interface ofthese two components. In some instances, after the first substrate isbonded to a second substrate, the structure may further be placed incontact with and bonded to a third substrate. In some instances, thethird substrate may be placed in contact with the side of the firstsubstrate that is not in contact with the second substrate. In someinstances, the first substrate is placed between the second substrateand the third substrate. In some instances, the second substrate and thethird substrate can cover and/or seal the grooves, indentations, orapertures formed on the first substrate to form the channels and/orchambers (e.g., the interior portion) of the device at the interface ofthese components.

The device can have openings that are oriented such that they are influid communication with at least one of the fluid channels and/or fluidchambers formed in the interior portion of the device, thereby formingfluid inlets and/or fluid outlets. In some instances, the openings areformed on the first substrate. In some instances, the openings areformed on the first and the second substrate. In some instances, theopenings are formed on the first, the second, and the third substrate.In some instances, the openings are positioned at the top side of thedevice. In some instances, the openings are positioned at the bottomside of the device. In some instances, the openings are positioned atthe first and/or the second ends of the device, and the channels runalong the direction from the first end to the second end.

Conditions under which substrates may be bonded together are generallywidely understood by those of skill in the art, and such bonding ofsubstrates is generally carried out by any of a variety of methods, thechoice of which may vary depending upon the nature of the substratematerials used. For example, thermal bonding of substrates may beapplied to a number of substrate materials including, e.g., glass orsilica-based substrates, as well as some polymer based-substrates. Suchthermal bonding techniques typically comprise mating the substratesurfaces that are to be bonded under conditions of elevated temperatureand, in some cases, application of external pressure. The precisetemperatures and pressures utilized will generally vary depending uponthe nature of the substrate materials used.

For example, for silica-based substrate materials, i.e., glass(borosilicate glass, PyrexTM soda lime glass, etc.), fused-silica(quartz), and the like, thermal bonding of substrates is typicallycarried out at temperatures ranging from about 500° C. to about 1400°C., and preferably, from about 500° C. to about 1200° C. For example,soda lime glass is typically bonded at temperatures of around 550° C.,whereas borosilicate glass is typically thermally bonded at or near 800°C. Quartz substrates, on the other hand, are typically thermally bondedat temperatures at or near 1200° C. These bonding temperatures aretypically achieved by placing the substrates to be bonded into hightemperature annealing ovens.

Polymeric substrates that are thermally bonded, on the other hand, willtypically utilize lower temperatures and/or pressures than silica-basedsubstrates, in order to prevent excessive melting of the substratesand/or distortion, e.g., flattening of the interior portion of thedevice (i.e., the fluid channels or chambers). Generally, such elevatedtemperatures for bonding polymeric substrates will vary from about 80°C. to about 200° C., depending upon the polymeric material used, andwill preferably be between about 90° C. and about 150° C. Because of thesignificantly reduced temperatures required for bonding polymericsubstrates, such bonding may typically be carried out without the needfor the high temperature ovens used in the bonding of silica-basedsubstrates. This allows incorporation of a heat source within a singleintegrated bonding system, as described in greater detail below.

Adhesives may also be used to bond substrates together according towell-known methods, which typically comprise applying a layer ofadhesive between the substrates that are to be bonded and pressing themtogether until the adhesive sets. A variety of adhesives may be used inaccordance with these methods, including, e.g., UV curable adhesives,that are commercially available. Alternative methods may also be used tobond substrates together in accordance with the present invention,including e.g., acoustic or ultrasonic welding and/or solvent welding ofpolymeric parts.

Typically, a number of the described microfluidic chips or devices willbe manufactured at the same time, e.g., using “wafer-scale” fabrication.For example, polymeric substrates may be stamped or molded in largeseparable sheets which can then be mated and bonded together. Individualdevices or bonded substrates may then be separated from the larger sheetby cutting or dicing. Similarly, for silica-based substrates, individualdevices can be fabricated from larger substrate wafers or plates,allowing higher throughput of the manufacturing process. Specifically, aplurality of fluid channel structures can be fabricated on a firstsubstrate wafer or plate, which is then overlaid with and bonded to asecond substrate wafer or plate, and optionally further overlaid withand bonded to a third substrate wafer or plate. The individual devicesare then segmented from the larger substrates using known methods, suchas sawing, scribing and breaking, and the like.

As noted above, the top or second substrate is overlaid upon the bottomor first substrate to seal the various channels and chambers. Incarrying out the bonding process according to the methods of the presentdisclosure, the bonding of the first and second substrates may becarried out using vacuum and/or pressure to maintain the two substratesurfaces in optimal contact. In particular, the bottom substrate may bemaintained in optimal contact with the top substrate by, e.g., matingthe planar surface of the bottom substrate with the planar surface ofthe top substrate and applying a vacuum through holes that are disposedthrough the top substrate. Typically, application of a vacuum to holesin the top substrate is carried out by placing the top substrate on avacuum chuck, which typically comprises a mounting table or surface,having an integrated vacuum source. In the case of silica-basedsubstrates, the bonded substrates are subjected to elevated temperaturesin order to create an initial bond, so that the bonded substrates maythen be transferred to the annealing oven, without any shifting relativeto each other.

Alternate bonding systems for incorporation with the apparatus describedherein include, e.g., adhesive dispensing systems, for applying adhesivelayers between the two planar surfaces of the substrates. This may bedone by applying the adhesive layer prior to mating the substrates, orby placing an amount of the adhesive at one edge of the adjoiningsubstrates and allowing the wicking action of the two mated substratesto draw the adhesive across the space between the two substrates.

In certain instances, the overall bonding system can include automatablesystems for placing the top and bottom substrates on the mountingsurface and aligning them for subsequent bonding. Typically, suchsystems include translation systems for moving either the mountingsurface or one or more of the top and bottom substrates relative to eachother. For example, robotic systems may be used to lift, translate andplace each of the top and bottom substrates upon the mounting table, andwithin the alignment structures, in turn. Following the bonding process,such systems also can remove the finished product from the mountingsurface and transfer these mated substrates to a subsequent operation,e.g., a separation or dicing operation, an annealing oven forsilica-based substrates, etc., prior to placing additional substratesthereon for bonding.

In some instances, the manufacturing of the microfluidic chip includesthe layering or laminating of two or more layers of substrate, e.g.,patterned and non-patterned polymeric sheets, in order to produce thechip. For example, in microfluidic devices, the microfluidic features ofthe device are typically produced by laser irradiation, etching, orotherwise fabricating features into the surface of a first layer. Asecond layer is then laminated or bonded to the surface of the first toseal these features and provide the fluidic elements of the device,e.g., the fluid channels.

As noted above, in some instances one or more capillary flow celldevices or microfluidic chips may be mounted in a cartridge chassis toform a capillary flow cell cartridge or microfluidic cartridge. In someinstances, the capillary flow cell cartridge or microfluidic cartridgemay further comprise additional components that are integrated with thecartridge to provide enhanced performance for specific applications.Examples of additional components that may be integrated into thecartridge include, but are not limited to, adapters or connectors formaking fluidic connections to other components of the system, fluid flowcontrol components (e.g., miniature valves, miniature pumps, mixingmanifolds, etc.), temperature control components (e.g., resistiveheating elements, metal plates that serve as heat sources or sinks,piezoelectric (Peltier) devices for heating or cooling, temperaturesensors), or optical components (e.g., optical lenses, windows, filters,mirrors, prisms, fiber optics, and/or light-emitting diodes (LEDs) orother miniature light sources that may collectively be used tofacilitate spectroscopic measurements and/or imaging of one or morecapillary or fluid flow channels.

The fluidic adaptors, cartridge chassis, and other cartridge componentsmay be attached to the capillaries, capillary flow cell devic(s),microfluidic chip(s) (or fluid channels within the chip) using any of avariety of techniques known to those of skill in the art including, butnot limited to, press fit, adhesive bonding, solvent bonding, laserwelding, etc., or any combination thereof. In some instances, theinlet(s) and/or outlet(s) of the microfluidic channels in themicrofluidic chip are apertures on the top surface of the chip, and thefluidic adaptors can be attached or coupled to the inlet(s) and/oroutlet(s) of the microfluidic channels within the chip. In someinstances, the cartridge may comprise additional adapters (i.e., inaddition to the fluidic adapters) that mate with the chip and/or fluidicadapters and help to position the chip within the cartridge. Theseadapters may be constructed using the same fabrication techniques andmaterials as those outlined above for the fluidic adapters.

The cartridge chassis (or “housing”) may be fabricated from metal and/orpolymer materials such as aluminum, anodized aluminum, polycarbonate(PC), acrylic (PMMA), or Ultem (PEI), while other materials are alsoconsistent with the present disclosure. A housing may be fabricatedusing CNC machining and/or molding techniques, and designed so that one,two, or more than two capillaries or microfluidic chips are constrainedby the chassis in a fixed orientation to create one or more independentflow channels. The capillaries or chips may be mounted in the chassisusing, e.g., a compression fit design, or by mating with compressibleadapters made of silicone or a fluoroelastomer. In some instances, twoor more components of the cartridge chassis (e.g., an upper half and alower half) are assembled using, e.g., screws, clips, clamps, or otherfasteners so that the two halves are separable. In some instances, twoor more components of the cartridge chassis are assembled using, e.g.,adhesives, solvent bonding, or laser welding so that the two or morecomponents are permanently attached.

Flow cell surface coatings: In some instances, one or more interiorsurfaces of the capillary lumens or microfluidic channels in thedisclosed flow cell devices may be coated using any of a variety ofsurface modification techniques or polymer coatings known to those ofskill in the art. In some instances, the coatings may be formulated toincrease or maximize the number of available binding sites (e.g.,tethered oligonucleotide adapter/primer sequences) on the one or moreinterior surfaces to increase or maximize a foreground signal, e.g., afluorescence signal arising from labeled nucleic acid moleculeshybridized to tethered oligonucleotide adapter/primer sequences. In someinstances, the coatings may be formulated to decrease or minimizenonspecific binding of fluorophores and other small molecules, orlabeled or unlabeled nucleotides, proteins, enzymes, antibodies,oligonucleotides, or nucleic acid molecules (e.g., DNA, RNA, etc.), inorder to decrease or minimize a background signal, e.g., backgroundfluorescence arising from the nonspecific binding of labeledbiomolecules or from autofluorescence of a sample support structure. Thecombination of increased foreground signal and reduced background signalthat may be achieved in some instances through the use of the disclosedcoatings may thus provide improved signal-to-noise ratio (SNR) inspectroscopic measurements or improved contrast-to-noise ratio (CNR) inimaging methods.

As will be discussed in more detail below, the disclosed hydrophilic,polymer-coated flow cell devices, optionally used in combination withthe improved hybridization and/or amplification protocols, yieldsolid-phase bioassay reactions that exhibit: (i) negligible non-specificbinding of protein and other reaction components (thus reducing orminimizing substrate background), (ii) negligible non-specific nucleicacid amplification product, and (iii) provide tunable nucleic acidamplification reactions. Although described herein primarily in thecontext of nucleic acid hybridization, amplification, and sequencingassays, it will be understood by those of skill in the art that thedisclosed low-binding supports may be used in any of a variety of otherbioassay formats including, but not limited to, sandwich immunoassays,enzyme-linked immunosorbent assays (ELISAs), etc.

In a preferred aspect, one or more layers of a coating material may beapplied to the interior flow cell device surfaces, where the number oflayers and/or the material composition of each layer is chosen to adjustone or more surface properties of the interior flow cell devicesurfaces, as noted in U.S. patent application Ser. No. 16/363,842.Examples of surface properties that may be adjusted include, but are notlimited to, surface hydrophilicity/hydrophobicity, overall coatingthickness, the surface density of chemically-reactive functional groups,the surface density of grafted linker molecules or oligonucleotideadapters/primers, etc. In some preferred applications, one or moresurface properties of the capillary or channel lumen are adjusted to,for example, (i) provide for very low non-specific binding of proteins,oligonucleotides, fluorophores, and other molecular components ofchemical or biological analysis applications, including solid-phasenucleic acid amplification and/or sequencing applications, (ii) providefor improved solid-phase nucleic acid hybridization specificity andefficiency, and (iii) provide for improved solid-phase nucleic acidamplification rate, specificity, and efficiency.

Any of a variety of molecules known to those of skill in the artincluding, but not limited to, silanes, amino acids, peptides,nucleotides, oligonucleotides, other monomers or polymers, orcombinations thereof may be used in creating the one or morechemically-modified layers on the interior flow cell device surfaces,where the choice of components used may be varied to alter one or moreproperties of the support surface, e.g., the surface density offunctional groups and/or tethered oligonucleotide primers, thehydrophilicity/hydrophobicity of the support surface, or the threethree-dimensional nature (i.e., “thickness”) of the support surface.

The attachment chemistry used to graft a first chemically-modified layerto an interior surface of the flow cell (capillary or channel) willgenerally be dependent on both the material from which the flow celldevice is fabricated and the chemical nature of the layer. In someinstances, the first layer may be covalently attached to the interiorflow cell device surfaces. In some instances, the first layer may benon-covalently attached, e.g., adsorbed to the surface throughnon-covalent interactions such as electrostatic interactions, hydrogenbonding, or van der Waals interactions between the surface and themolecular components of the first layer. In either case, the substratesurface may be treated prior to attachment or deposition of the firstlayer. Any of a variety of surface preparation techniques known to thoseof skill in the art may be used to clean or treat the support surface.For example, glass or silicon surfaces may be acid-washed using aPiranha solution (a mixture of sulfuric acid (H₂SO₄) and hydrogenperoxide (H₂O₂)) and/or cleaned using an oxygen plasma treatment method.

Silane chemistries constitute one non-limiting approach for covalentlymodifying the silanol groups on glass or silicon surfaces to attach morereactive functional groups (e.g., amines or carboxyl groups), which maythen be used in coupling linker molecules (e.g., linear hydrocarbonmolecules of various lengths, such as C6, C12, C18 hydrocarbons, orlinear polyethylene glycol (PEG) molecules) or layer molecules (e.g.,branched PEG molecules or other polymers) to the surface. Examples ofsuitable silanes that may be used in creating any of the disclosed lowbinding support surfaces include, but are not limited to,(3-Aminopropyl) trimethoxysilane (APTMS), (3-Aminopropyl)triethoxysilane (APTES), any of a variety of PEG-silanes (e.g.,comprising molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEGsilane (i.e., comprising a free amino functional group), maleimide-PEGsilane, biotin-PEG silane, and the like.

Examples of preferred polymers that may be used to create one or morelayers of low non-specific binding material in any of the disclosedsupport surfaces include, but are not limited to, polyethylene glycol(PEG) of various molecular weights and branching structures,streptavidin, polyacrylamide, polyester, dextran, poly-lysine, andpoly-lysine copolymers, or any combination thereof. Examples ofconjugation chemistries that may be used to graft one or more layers ofmaterial (e.g. polymer layers) to the support surface and/or tocross-link the layers to each other include, but are not limited to,biotin-streptavidin interactions (or variations thereof), His tag—Ni/NTAconjugation chemistries, methoxy ether conjugation chemistries,carboxylate conjugation chemistries, amine conjugation chemistries, NHSesters, maleimides, thiol, epoxy, azide, hydrazide, alkyne, isocyanate,and silane.

In some instances, the number of layers of polymer or other chemicallayers on the interior flow cell device surfaces may range from 1 toabout 10, or greater than 10. In some instances, the number of layers isat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, or at least 10. In some instances,the number of layers may be at most 10, at most 9, at most 8, at most 7,at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the number of layers may range from about 2to about 4. In some instances, the one or more layers may all comprisethe same material. In some instances, each layer may comprise adifferent material. In some instances, a plurality of layers maycomprise a plurality of materials.

One or more layers of a multi-layered surface may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(-hydroxylethyl methacrylate)(branced PHEMA), branched poly(oligo(ethylene glycol) methyl ethermethacrylate) (branched POEGMA), branched polyglutamic acid (branchedPGA), branched poly-lysine, branched poly-glucoside, and dextran.

In some instances, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branches. Molecules often exhibit a ‘power of 2’ number of branches,such as 2, 4, 8, 16, 32, 64, or 128 branches.

In some instances, the resulting functional end groups distal from thesurface following the deposition of one or more layers, e.g., polymerlayers can include, but are not limited to, biotin, methoxy ether,carboxylate, amine, NHS ester, maleimide, and bis-silane.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500 Daltons, at least 1,000 Daltons, atleast 1,500 Daltons, at least 2,000 Daltons, at least 2,500 Daltons, atleast 3,000 Daltons, at least 3,500 Daltons, at least 4,000 Daltons, atleast 4,500 Daltons, at least 5,000 Daltons, at least 7,500 Daltons, atleast 10,000 Daltons, at least 12,500 Daltons, at least 15,000 Daltons,at least 17,500 Daltons, at least 20,000 Daltons, at least 25,000Daltons, at least 30,000 Daltons, at least 35,000 Daltons, at least40,000 Daltons, at least 45,000 Daltons, or at least 50,000 Daltons. Insome instances, the linear, branched, or multi-branched polymers used tocreate one or more layers of any of the multi-layered surfaces disclosedherein may have a molecular weight of at most 50,000 Daltons, at most45,000 Daltons, at most 40,000 Daltons, at most 35,000 Daltons, at most30,000 Daltons, at most 25,000 Daltons, at most 20,000 Daltons, at most17,500 Daltons, at most 15,000 Daltons, at most 12,500 Daltons, at most10,000 Daltons, at most 7,500 Daltons, at most 5,000 Daltons, at most4,500 Daltons, at most 4,000 Daltons, at most 3,500 Daltons, at most3,000 Daltons, at most 2,500 Daltons, at most 2,000 Daltons, at most1,500 Daltons, at most 1,000 Daltons, or at most 500 Daltons. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the molecular weight of linear, branched, ormulti-branched polymers used to create one or more layers of any of themulti-layered surfaces disclosed herein may range from about 1,500Daltons to about 20,000 Daltons. Those of skill in the art willrecognize that the molecular weight of linear, branched, ormulti-branched polymers used to create one or more layers of any of themulti-layered surfaces disclosed herein may have any value within thisrange, e.g., about 1,260 Daltons.

In some instances, two or more layers may be covalently coupled to eachother or internally cross-linked to improve the stability of theresulting surface. In some instances, e.g., wherein at least one layerof a multi-layered surface comprises a branched polymer, the number ofcovalent bonds between a branched polymer molecule of the layer beingdeposited and molecules of the previous layer may range from about onecovalent linkage per molecule and about 32 covalent linkages permolecule. In some instances, the number of covalent bonds between abranched polymer molecule of the new layer and molecules of the previouslayer may be at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least12, at least 14, at least 16, at least 18, at least 20, at least 22, atleast 24, at least 26, at least 28, at least 30, or at least 32, or morethan 32 covalent linkages per molecule. In some instances, the number ofcovalent bonds between a branched polymer molecule of the new layer andmolecules of the previous layer may be at most 32, at most 30, at most28, at most 26, at most 24, at most 22, at most 20, at most 18, at most16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7,at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the number of covalent bonds between abranched polymer molecule of the new layer and molecules of the previouslayer may range from about 4 to about 16. Those of skill in the art willrecognize that the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the previous layer may haveany value within this range, e.g., about 11 in some instances, or anaverage number of about 4.6 in other instances.

Any reactive functional groups that remain following the coupling of amaterial layer to the interior flow cell device surfaces may optionallybe blocked by coupling a small, inert molecule using a high yieldcoupling chemistry. For example, in the case that amine couplingchemistry is used to attach a new material layer to the previous one,any residual amine groups may subsequently be acetylated or deactivatedby coupling with a small amino acid such as glycine.

In order to scale binding site surface density, e.g., oligonucleotideadapter/primer surface density, and add additional dimensionality tohydrophilic or amphoteric surfaces, substrates comprising multi-layercoatings of PEG and other hydrophilic polymers have been developed. Byusing hydrophilic and amphoteric surface layering approaches thatinclude, but are not limited to, the polymer/co-polymer materialsdescribed below, it is possible to increase adapter/primer loadingdensity on the surface significantly. Traditional PEG coating approachesuse monolayer primer deposition, which has been tested and reported forsingle molecule sequencing applications but do not yield high copynumbers for nucleic acid amplification applications. As describedherein, “layering” can be accomplished using traditional crosslinkingapproaches with any compatible polymer or monomer subunits such that asurface comprising two or more highly crosslinked layers can be builtsequentially. Examples of suitable polymers include, but are not limitedto, streptavidin, polyacrylamide, polyester, dextran, poly-lysine, andcopolymers of poly-lysine and PEG. In some instances, the differentlayers may be cross-linked to each other through any of a variety ofconjugation reactions including, but not limited to, biotin-streptavidinbinding, azide-alkyne click reaction, amine-NHS ester reaction,thiol-maleimide reaction, and ionic interactions between positivelycharged polymer and negatively charged polymer. In some instances, highadapter/primer density materials may be constructed in solution andsubsequently layered onto the surface in multiple steps.

Exemplary PEG multilayers include PEG (8 arm, 16 arm, 8 arm) onPEG-amine-APTES. Similar concentrations were observed for 3-layermulti-arm PEG (8 arm,16 arm, 8arm) and (8arm, 64arm, 8arm) onPEG-amine-APTES exposed to 8uM primer, and 3-layer multi-arm PEG (8arm,8arm, 8arm) using star-shape PEG-amine to replace 16 arm and 64 arm. PEGmultilayers having comparable first, second and third PEG layers arealso contemplated.

In some instances, the resultant surface density of binding sites on theinterior flow cell device surfaces, e.g., oligonucleotide adapter/primersurface densities, may range from about 100 primer molecules per μm² toabout 1,000,000 primer molecules per μm². In some instances, the surfacedensity of binding sites may be at least 100, at least 200, at least300, at least 400, at least 500, at least 600, at least 700, at least800, at least 900, at least 1,000, at least 1,500, at least 2,000, atleast 2,500, at least 3,000, at least 3,500, at least 4,000, at least4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500,at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least9,000, at least 9,500, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, or at least 1,000,000 molecules per μm². Insome instances, the surface density of binding sites may be at most1,000,000, at most 950,000, at most 900,000, at most 850,000, at most800,000, at most 750,000, at most 700,000, at most 650,000, at most600,000, at most 550,000, at most 500,000, at most 450,000, at most400,000, at most 350,000, at most 300,000, at most 250,000, at most200,000, at most 150,000, at most 100,000, at most 95,000, at most90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000,at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000,at most 20,000, at most 15,000, at most 10,000, at most 9,500, at most9,000, at most 8,500, at most 8,000, at most 7,500, at most 7,000, atmost 6,500, at most 6,000, at most 5,500, at most 5,000, at most 4,500,at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most700, at most 600, at most 500, at most 400, at most 300, at most 200, orat most 100 molecules per pm². Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thesurface density of binding sites may range from about 10,000 moleculesper pm² to about 100,000 molecules per pm². Those of skill in the artwill recognize that the surface density of binding sites may have anyvalue within this range, e.g., about 3,800 molecules per pm² in someinstances, or about 455,000 molecules per pm² in other instances. Insome instances, as will be discussed further below for nucleic acidsequencing applications, the surface density of template library nucleicacid sequences (e.g., sample DNA molecules) initially hybridized toadapter or primer sequences tethered to the interior flow cell devicesurfaces may be less than or equal to that indicated for the surfacedensity of binding sites. In some instances, as will also be discussedfurther below, the surface density of clonally-amplified templatelibrary nucleic acid sequences hybridized to adapter or primer sequenceson the interior flow cell device surfaces may span the same range or adifferent range as that indicated for the surface density of tetheredoligonucleotide adapters or primers.

Local surface densities of binding sites on the interior flow celldevice surfaces as listed above do not preclude variation in densityacross a surface, such that a surface may comprise a region having abinding site density of, for example, 500,000/um², while also comprisingat least a second region having a substantially different local surfacedensity.

In some instances, capture probes, e.g., oligonucleotide primers withdifferent base sequences and base modifications (or other biomolecules,e.g., enzymes or antibodies) may be tethered to oneor more layers of theresulting surface at various surface densities. In some instances, forexample, both surface functional group density and the capture probeconcentration used for coupling may be varied to target a certaincapture probe surface density range. Additionally, capture probe surfacedensity may be controlled by diluting capture probes with other “inert”molecules that carry the same reactive functional group for coupling tothe surface. For example, amine-labeled oligonucleotide probes can bediluted with amine-labeled polyethylene glycol in a reaction with anNHS-ester coated surface to reduce the final primer density. In the caseof oligonucleotide adapters/primers, probe sequences with differentlengths of linker between the hybridization region and the surfaceattachment functional group may also be applied to vary surface density.Example of suitable linkers include poly-T and poly-A strands at the 5′end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20monomer units), and carbon-chain (e.g., C6, C12, C18, etc.). To measureor estimate the capture probe surface density, fluorescently labeledcapture probes may be tethered to the surface and a fluorescence readingthen compared with that for a calibration solution comprising a knownconcentration of the fluorophore.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces, e.g., interiorflow cell device surfaces, may be assessed, for example, through themeasurement of water contact angles in which a small droplet of water isplaced on the surface and its angle of contact with the surface ismeasured using, e.g., an optical tensiometer. In some instances, astatic contact angle may be determined. In some instances, an advancingor receding contact angle may be determined. In some instances, thewater contact angle for the hydrophilic, low-binding support surfaceddisclosed herein may range from about 0 degrees to about 50 degrees. Insome instances, the water contact angle for the hydrophilic, low-bindingsupport surfaced disclosed herein may no more than 50 degrees, 45degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, 18degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6degrees, 4 degrees, 2 degrees, or 1 degree. In many cases the contactangle is no more than any value within this range, e.g., no more than 40degrees. Those of skill in the art will realize that a givenhydrophilic, low-binding support surface of the present disclosure mayexhibit a water contact angle having a value of anywhere within thisrange, e.g., about 27 degrees. In some instances, the disclosed lownonspecific binding surfaces have a water contact angle of less than 45degrees. In some instances, the disclosed low nonspecific bindingsurfaces have a water contact angle of less than 35 degrees.

As noted, the hydrophilic coated interior flow cell device surfaces ofthe present disclosure exhibit reduce non-specific binding of proteins,nucleic acids, fluorophores, and other components of biological andbiochemical assay methods. The degree of non-specific binding exhibitedby a given support surface, e.g., an interior flow cell device surface,may be assessed either qualitatively or quantitatively. For example, insome instances, exposure of the surface to fluorescent dyes (e.g.,cyanine dye 3 (Cy3), cyanine dye 5 (Cy5), etc.), fluorescently-labelednucleotides, fluorescently-labeled oligonucleotides, and/orfluorescently-labeled proteins (e.g. polymerases) under a standardizedset of conditions, followed by a specified rinse protocol andfluorescence imaging may be used as a qualitative tool for comparison ofnon-specific binding on supports comprising different surfaceformulations. In some instances, exposure of the surface to fluorescentdyes, fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, and/or fluorescently-labeled proteins (e.g.polymerases) under a standardized set of conditions, followed by aspecified rinse protocol and fluorescence imaging may be used as aquantitative tool for comparison of non-specific binding on supportscomprising different surface formulations—provided that care has beentaken to ensure that the fluorescence imaging is performed underconditions where fluorescence signal is linearly related (or related ina predictable manner) to the number of fluorophores on the supportsurface (e.g., under conditions where signal saturation and/orself-quenching of the fluorophore is not an issue) and suitablecalibration standards are used. In some instances, other techniquesknown to those of skill in the art, for example, radioisotope labelingand counting methods may be used for quantitative assessment of thedegree to which non-specific binding is exhibited by the differentsupport surface formulations of the present disclosure.

In some instances, the degree of non-specific binding exhibited by thedisclosed low nonspecific binding support surfaces may be assessed usinga standardized protocol for contacting the surface with a labeledprotein (e.g., bovine serum albumin (BSA), streptavidin, a DNApolymerase, a reverse transcriptase, a helicase, a single-strandedbinding protein (SSB), etc., or any combination thereof), a labelednucleotide, a labeled oligonucleotide, etc., under a standardized set ofincubation and rinse conditions, followed be detection of the amount oflabel remaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label known to one of skill in the art. In someinstances, the degree of non-specific binding exhibited by a givensupport surface formulation may thus be assessed in terms of the numberof non-specifically bound protein molecules (or other molecules) perunit area. In some instances, the low nonspecific binding supports ofthe present disclosure may exhibit nonspecific protein binding (ornonspecific binding of other specified molecules, e.g., cyanine dye 3(Cy3) of less than 0.001 molecule per μm², less than 0.01 molecule perμm², less than 0.1 molecule per μm², less than 0.25 molecule per μm²,less than 0.5 molecule per μm², less than 1 molecule per μm², less than10 molecules per μm², less than 100 molecules per μm², or less than1,000 molecules per μm². Those of skill in the art will realize that agiven support surface of the present disclosure may exhibit nonspecificbinding falling anywhere within this range, for example, of less than 86molecules per pm². For example, some modified surfaces disclosed hereinexhibit nonspecific protein binding of less than 0.5 molecule/pm²following contact with a 1 uM solution of Cy3 labeled streptavidin (GEAmersham) in phosphate buffered saline (PBS) buffer for 15 minutes,followed by 3 rinses with deionized water. Some modified surfacesdisclosed herein exhibit nonspecific binding of Cy3 dye molecules ofless than 0.25 molecules per p.m2. In independent nonspecific bindingassays, 1μM labeled Cy3 SA (ThermoFisher), 1 uM Cy5 SA dye(ThermoFisher), 10 uM Aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10uM Aminoallyl-dUTP-ATTO-Rholl (Jena Biosciences), 10 uM Aminoallyl-dUTP-ATTO-Rholl (Jena Biosciences), 10 uM 7-Propargylamino-7-deaza-dGTP-Cy5(Jena Biosciences, and 10 uM 7-Propargylamino-7-deaza-dGTP-Cy3 (JenaBiosciences) were incubated on the low nonspecific binding substrates at37° C. for 15 minutes in a 384 well plate format. Each well was rinsed2-3 x with 50 ul deionized RNase/DNase Free water and 2-3 x with 25 mMACES buffer pH 7.4. The 384 well plates were imaged on a GE Typhoon(GEHealthcare Lifesciences, Pittsburgh, PA) instrument using the Cy3,AF555, or Cy5 filter sets (according to dye test performed) as specifiedby the manufacturer at a PMT gain setting of 800 and resolution of50-100 μm. For higher resolution imaging, images were collected on anOlympus IX83 microscope (Olympus Corp., Center Valley, Pa.) with a totalinternal reflectance fluorescence (TIRF) objective (20×, 0.75 NA or100X, 1.5 NA, Olympus), an sCMOS Andor camera (Zyla 4.2. Dichroicmirrors were purchased from Semrock (IDEX Health & Science, LLC,Rochester, New York), e.g., 405, 488, 532, or 633 nm dichroicreflectors/beamsplitters, and band pass filters were chosen as 532 LP or645 LP concordant with the appropriate excitation wavelength. Somemodified surfaces disclosed herein exhibit nonspecific binding of dyemolecules of less than 0.25 molecules per pm².

In some instances, the coated flow cell device surfaces disclosed hereinmay exhibit a ratio of specific to nonspecific binding of a fluorophoresuch as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, orany intermediate value spanned by the range herein.

In some instances, one or more surface modification and/or polymerlayers may be applied to the interior flow cell device surfaces using atechnique such as chemical vapor deposition (CVD). In some instances,one or more surface modification and/or polymer layers may be applied tothe interior flow cell device surfaces by flowing one or moreappropriate chemical coupling or coating reagents through thecapillaries or fluid channels prior to use for their intendedapplication. In some instances, one or more coating reagents may beadded to a buffer used, e.g., a nucleic acid hybridization,amplification reaction, and/or sequencing reaction buffer to provide fordynamic coating of the interior flow cell device surfaces.

In some instances, the chemical modification layers may be applieduniformly across the surface of the substrate or support structure.Alternately, the surface of the substrate or support structure may benon-uniformly distributed or patterned, such that the chemicalmodification layers are confined to one or more discrete regions of thesubstrate. For example, the substrate surface may be patterned usingphotolithographic techniques to create an ordered array or randompattern of chemically-modified regions on the surface. Alternately or incombination, the substrate surface may be patterned using, e.g., contactprinting and/or ink-jet printing techniques. In some instances, anordered array or random patter of chemically-modified discrete regionsmay comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, or 10,000 or more discrete regions, or anyintermediate number of discrete regions spanned by the range herein.

In some instances, fluorescence images of the disclosed low nonspecificbinding surfaces when used, e.g., in nucleic acid hybridization oramplification applications to create clusters of hybridized orclonally-amplified nucleic acid molecules (e.g., “discrete regions” thathave been directly or indirectly labeled with a fluorophore) exhibitcontrast-to-noise ratios (CNRs) of at least 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210,220, 230, 240, 250, or greater than 250 when the nucleic acid moleculesare labeled with Cy3 and the images are acquired using an Olympus IX83inverted fluorescence microscope equipped with a 20×, 0.75 NA objective,a 532 nm light source, a bandpass and dichroic mirror filter set adaptedor optimized for 532 nm long-pass excitation and Cy3 fluorescenceemission filter, a Semrock 532 nm dichroic reflector, and a camera(Andor sCMOS, Zyla 4.2) where the excitation light intensity is adjustedto avoid signal saturation, and the surface is immersed in a buffer(e.g., 25 mM ACES, pH 7.4 buffer) while the image is acquired. As usedherein, contrast-to-noise ratio (CNR) is calculated as:

CNR=(S−B)/Noise

where S=foreground signal (e.g., the fluorescence signal as measured inthe image that arises from a labeled nucleic acid colony or cluster on asample support surface), B=background signal (where B=B inter+B intra),B inter=background signal measured at a location on the sample supportsurface that is between labeled nucleic acid colonies or clusters, Bintra=background signal measured at the location of a nucleic acidcolony or cluster (determined, e.g., by contacting the sample supportsurface with a labeled, non-complementary oligonucleotide and measuringthe resulting fluorescence), and Noise=the signal noise. Thecontrast-to-noise ratio (CNR) of images of sequencing surfaces, forexample, provides a key metric in assessing nucleic acid amplificationspecificity and non-specific binding on the support. Whilesignal-to-noise ratio (SNR) is often considered to be a benchmark ofoverall signal quality, it can be shown that improved CNR can provide asignificant advantage over SNR as a benchmark for signal quality inimaging applications that require rapid image capture (e.g., nucleicacid sequencing applications for which cycle times must be minimized).

In some instances, polymer-coated sample support structures, e.g.,interior flow cell device surfaces comprising the disclosed hydrophilicpolymer coatings, may exhibit improved stability to repetitive exposureto solvents, changes in temperature, changes in pH, or long-termstorage.

Fluidics systems and fluid flow control modules: in someimplementations, the disclosed imaging and/or analysis systems mayprovide fluid flow control capability for delivering samples or reagentsto the one or more flow cell devices or flow cell cartridges (e.g.,single capillary flow cell device or microfluidic channel flow celldevice) connected to the system. Reagents and buffers may be stored inbottles, reagent and buffer cartridges, or other suitable containersthat are connected to the flow cell inlets by means of tubing and valvemanifolds. The disclosed systems may also include processed sample andwaste reservoirs in the form of bottles, cartridges, or other suitablecontainers for collecting fluids downstream of the capillary flow celldevices or capillary flow cell cartridges. In some embodiments, thefluid flow (or “fluidics”) control module may provide programmableswitching of flow between different sources, e.g. sample or reagentreservoirs or bottles located in the instrument, and the inlet(s) to acentral region (e.g., a capillary flow cell or microfluidic device, or alarge fluid chamber such as a large fluid chamber within a microfluidicdevice). In some instances, the fluid flow control module may provideprogrammable switching of flow between outlet(s) from the central region(e.g., a capillary flow cell or microfluidic device) and differentcollection points, e.g., processed sample reservoirs, waste reservoirs,etc., connected to the system. In some instances, samples, reagents,and/or buffers may be stored within reservoirs that are integrated intothe flow cell cartridge or microfluidic cartridge itself. In someinstances, processed samples, spent reagents, and/or used buffers may bestored within reservoirs that are integrated into the flow cellcartridge or microfluidic device cartridge itself

In some implementations, one or more fluid flow control modules may beconfigured to control the delivery of fluids to one or more capillaryflow cells, capillary flow cell cartridges, microfluidic devices,microfluidic cartridges, or any combination thereof. In some instances,the one or more fluidics controllers may be configured to controlvolumetric flow rates for one or more fluids or reagents, linear flowvelocities for one or more fluids or reagents, mixing ratios for one ormore fluids or reagents, or any combination thereof. Control of fluidflow through the disclosed systems will typically be performed usingpumps (or other fluid actuation mechanisms) and valves (e.g.,programmable pumps and valves). Examples of suitable pumps include, butare not limited to, syringe pumps, programmable syringe pumps,peristaltic pumps, diaphragm pumps, and the like. Examples of suitablevalves include, but are not limited to, check valves, electromechanicaltwo-way or three-way valves, pneumatic two-way and three-way valves, andthe like. In some instances, fluid flow through the system may becontrolled by means of applying positive pneumatic pressure to one ormore inlets of the reagent and buffer containers, or to inletsincorporated into flow cell cartridge(s) (e.g., capillary flow cell ormicrofluidic cartridges). In some embodiments, fluid flow through thesystem may be controlled by means of drawing a vacuum at one or moreoutlets of waste reservoir(s), or at one or more outlets incorporatedinto flow cell cartridge(s) (e.g., capillary flow cell or microfluidiccartridges).

In some instances, different modes of fluid flow control are utilized atdifferent points in an assay or analysis procedure, e.g. forward flow(relative to the inlet and outlet for a given capillary flow celldevice), reverse flow, oscillating or pulsatile flow, or combinationsthereof. In some applications, oscillating or pulsatile flow may beapplied, for example, during assay wash/rinse steps to facilitatecomplete and efficient exchange of fluids within the one or more flowcell devices or flow cell cartridges (e.g., capillary flow cell devicesor cartridges, and microfluidic devices or cartridges).

Similarly, in some cases different fluid flow rates may be utilized atdifferent locations within a flow cell device or at different points inthe assay or analysis process workflow, for example, in some instances,the volumetric flow rate may vary from −100 ml/sec to +100 ml/sec. Insome embodiment, the absolute value of the volumetric flow rate may beat least 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, atleast 1 ml/sec, at least 10 ml/sec, or at least 100 ml/sec. In someembodiments, the absolute value of the volumetric flow rate may be atmost 100 ml/sec, at most 10 ml/sec, at most 1 ml/sec, at most 0.1ml/sec, at most 0.01 ml/sec, or at most 0.001 ml/sec. The volumetricflow rate at a given location with the flow cell device or at a givenpoint in time may have any value within this range, e.g. a forward flowrate of 2.5 ml/sec, a reverse flow rate of -0.05 ml/sec, or a value of 0ml/sec (i.e., stopped flow).

In some implementations, the fluidics system may be designed to minimizethe consumption of key reagents (e.g., expensive reagents) required forperforming, e.g., genomic analysis applications. For example, in someimplementations the disclosed fluidics systems may comprise a firstreservoir housing a first reagent or solution, a second reservoirhousing a second reagent or solution, and a central region, e.g., acentral capillary flow cell or microfluidic device, where an outlet fromthe first reservoir and an outlet from the second reservoir arefluidically coupled to an inlet of the central capillary flow cell ormicrofluidic device through at least one valve such that the volume ofthe first reagent or solution flowing per unit time from the outlet ofthe first reservoir to the inlet of the central capillary flow cell ormicrofluidic device is less than the volume of the second reagent orsolution flowing per unit time from the outlet of the second reservoirto the inlet of the central region. In some implementations, the firstreservoir and second reservoir may be integrated into a capillary flowcell cartridge or microfluidic cartridge. In some instances, the atleast one valve may also be integrated into the capillary flow cellcartridge or microfluidic cartridge.

In some instances, the first reservoir is fluidically coupled to thecentral capillary flow cell or microfluidic device through a firstvalve, and the second reservoir is fluidically coupled to the centralcapillary flow cell or microfluidic device through a second valve. Insome instances, the first and/or second valves may be, e.g., a diaphragmvalve, pinch valve, gate valve, or other suitable valve. In someinstances, the first reservoir is positioned in close proximity to theinlet of the central capillary flow cell or microfluidic device toreduce dead volume for delivery of the first reagent solution. In someinstances, the first reservoir is placed in closer proximity to theinlet of the central capillary flow cell or microfluidic device than isthe second reservoir. In some instances, the first reservoir ispositioned in close proximity to the second valve so as to reduce thedead volume for delivery of the first reagent relative to that fordelivery of a plurality of “second” reagents (e.g., two, three, four,five, or six or more “second” reagents) from a plurality of “second”reservoirs (e.g., two, three, four, five, or six or more “second”reservoirs).

The first and second reservoirs described above may be used to house thesame or different reagents or solutions. In some instances, the firstreagent that is housed in the first reservoir is different from thesecond reagent that is housed in the second reservoir, and the secondreagent comprises at least one reagent that is used in common by aplurality of reactions occurring in the central a central capillary flowcell or microfluidic device. In some instances, e.g., in fluidicssystems configured for performing nucleic acid sequencing chemistrywithin the central capillary flow cell or microfluidic device, the firstreagent comprises at least one reagent selected from the groupconsisting of a polymerase, nucleotide, and a nucleotide analog. In someinstances, the second reagent comprises a low-cost reagent, e.g., asolvent.

In some instances, the interior volume of the central region, e.g., acentral capillary flow cell cartridge, or microfluidic device comprisingone or more fluid channels or fluid chambers, can be adjusted based onthe specific application to be performed, e.g., nucleic acid sequencing.

In some embodiments, the central region comprises an interior volumesuitable for sequencing a eukaryotic genome. In some embodiments, thecentral region comprises an interior volume suitable for sequencing aprokaryotic genome. In some embodiments, the central region comprises aninterior volume suitable for sequencing a viral genome. In someembodiments, the central region comprises an interior volume suitablefor sequencing a transcriptome. For example, in some embodiments, theinterior volume of the central region may comprise a volume of less than0.05 μl, between 0.05 μl and 0.1 μl, between 0.05 μl and 0.2 μl, between0.05 μl and 0.5 μl, between 0.05 μl and 0.8 μl, between 0.05 μl and 1μl,between 0.05 μl and 1.2 μl, between 0.05 μl and 1.5 μl, between 0.1 μland 1.5 μl, between 0.2 μl and 1.5 μl, between 0.5 μl and 1.5 μl,between 0.8 μl and 1.5 μl, between 1μl and 1.5 μl, between 1.2 μl and1.5 μl, or greater than 1.5 μl, or a range defined by any two of theforegoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 0.5 μl, between 0.5 μl and1μl, between 0.5 μl and 2μl, between 0.5 μl and 5μl, between 0.5 μl and8μl, between 0.5 μl and 10 μl, between 0.5 μl and 12 μl, between 0.5 μland 15 μl, between 1 μl and 15 μl, between 2μl and 15 μl, between 5μland 15 μl, between 8μl and 15 μl, between 10 μl and 15 μl, between 12 μland 15 μl, or greater than 15 μl, or a range defined by any two of theforegoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 5 μl, between 5μl and 10 μl,between 5μl and 20 μl, between 5μl and 500 μl, between 5μl and 80 μl,between 5μl and 100 μl, between 5μl and 120 μl, between 5μl and 150 μl,between 10 μl and 150 μl, between 20 μl and 150 μl, between 50 μl and150 μl, between 80 μl and 150 μl, between 100 μl and 150 μl, between 120μl and 150 μl, or greater than 150 μl, or a range defined by any two ofthe foregoing. In some embodiments, the interior volume of the centralregion may comprise a volume of less than 50 μl, between 50 μl and 100μl, between 50 μl and 200 μl, between 50 μl and 500 μl, between 50 μland 800 μl, between 50 μl and 1000 μl, between 50 μl and 1200 μl,between 50 μl and 1500 μl, between 100 μl and 1500 μl, between 200 μland 1500 μl, between 500 μl and 1500 μl, between 800 μl and 1500 μl,between 1000 μl and 1500 μl, between 1200 μl and 1500 μl, or greaterthan 1500 μl, or a range defined by any two of the foregoing. In someembodiments, the interior volume of the central region may comprise avolume of less than 500 μl, between 500 μl and 1000 μl, between 500 μland 2000 μl, between 500 μl and 5 ml, between 500 μl and 8 ml, between500 μl and 10 ml, between 500 μl and 12 ml, between 500 μl and 15 ml,between 1 ml and 15 ml, between 2 ml and 15 ml, between 5 ml and 15 ml,between 8 ml and 15 ml, between 10m1 and 15 ml, between 12 ml and 15 ml,or greater than 15 ml, or a range defined by any two of the foregoing.In some embodiments, the interior volume of the central region maycomprise a volume of less than 5 ml, between 5 ml and 10 ml, between 5ml and 20 ml, between 5 ml and 50 ml, between 5 ml and 80 ml, between 5ml and 100 ml, between 5 ml and 120 ml, between 5 ml and 150 ml, between10 ml and 150 ml, between 20 ml and 150 ml, between 50 ml and 150 ml,between 80 ml and 150 ml, between 100m1 and 150 ml, between 120 ml and150 ml, or greater than 150 ml, or a range defined by any two of theforegoing. In some embodiments, the systems described herein comprise anarray or collection of flow cell devices or systems comprising multiplediscrete capillaries, microfluidic channels, fluidic channels, chambers,or lumenal regions, wherein the combined interior volume is, comprises,or includes one or more of the values within a range disclosed herein.

In some instances, the ratio of volumetric flow rate for the delivery ofthe first reagent to the central capillary flow cell or microfluidicdevice to that for delivery of the second reagent to the centralcapillary flow cell or microfluidic device may be less than 1:20, lessthan 1:16, least than 1:12, less than 1:10, less than 1:8, less than1:6, or less than 1:2. In some instances, the ratio of volumetric flowrate for the delivery of the first reagent to the central capillary flowcell or microfluidic device to that for delivery of the second reagentto the central capillary flow cell or microfluidic device may have anyvalue with the range spanned by these values, e.g., less than 1:15.

As noted, the flow cell devices and/or fluidics systems disclosed hereinmay be configured to achieve a more efficient use of the reagents thanthat achieved by, e.g., other sequencing devices and systems,particularly for the costly reagents used in a variety of sequencingchemistry steps. In some instances, the first reagent comprises areagent that is more expensive than the second reagent. In someinstances, the first reagent comprises a reaction-specific reagent andthe second reagent comprises a nonspecific reagent common to allreactions performed in the central capillary flow cell or microfluidicdevice region, and wherein the reaction specific reagent is moreexpensive than the nonspecific reagent.

In some instances, utilization of the flow cell devices and/or fluidicsystems disclosed herein may convey advantages in terms of reducedconsumption of costly reagents. In some instances, for example,utilization of the flow cell devices and/or fluidic systems disclosedherein may results in at least a 5%, at least a 7.5%, at least a 10%, atleast a 12.5%, at least a 15%, at least a 17.5%, at least a 20%, atleast a 22.5%, at least a 25%, at least a 30%, at least a 35%, at leasta 40%, at least a 45%, or at least a 50% reduction in reagentconsumption compared to the reagent consumption encountered whenoperating, e.g., current commercially-available nucleic acid sequencingsystems.

FIG. 31 illustrates a non-limiting example of a simple fluidics systemcomprising a single capillary flow cell connected to various fluid flowcontrol components, where the single capillary is optically accessibleand compatible with mounting on a microscope stage or in a customimaging instrument for use in various imaging applications. A pluralityof reagent reservoirs is fluidically-coupled with the inlet end of thesingle capillary flow cell device, where the reagent flowing through thecapillary at any given point in time is controlled by means of aprogrammable rotary valve that allows the user to control the timing andduration of reagent flow. In this non-limiting example, fluid flow iscontrolled by means of a programmable syringe pump that provides precisecontrol and timing of volumetric fluid flow and fluid flow velocity.

FIG. 32 illustrates a non-limiting example of a fluidics system thatcomprises a capillary flow cell cartridge having integrated diaphragmvalves to reduce or minimize dead volume and conserve certain keyreagents. The integration of miniature diaphragm valves into thecartridge allows the valve to be positioned in close proximity to theinlet of the capillary, thereby reducing or minimizing dead volumewithin the device and reducing the consumption of costly reagents. Theintegration of valves and other fluid control components within thecapillary flow cell cartridge also allows greater fluid flow controlfunctionality to be incorporated into the cartridge design.

FIG. 33 illustrates a non-limiting example of a capillary flow cellcartridge-based fluidics system used in combination with a microscopesetup, where the cartridge incorporates or mates with a temperaturecontrol component such as a metal plate that makes contact with thecapillaries within the cartridge and serves as a heat source/sink. Themicroscope setup consists of an illumination system (e.g., including alaser, LED, or halogen lamp, etc., as a light source), an objectivelens, an imaging system (e.g., a CMOS or CCD camera), and a translationstage to move the cartridge relative to the optical system, whichallows, e.g., fluorescence and/or bright field images to be acquired fordifferent regions of the capillary flow cells as the stage is moved.

Temperature control modules: In some implementations the disclosedsystems will include temperature control functionality for the purposeof facilitating the accuracy and reproducibility of assay or analysisresults. Examples of temperature control components that may beincorporated into the instrument system (or capillary flow cellcartridge) design include, but are not limited to, resistive heatingelements, infrared light sources, Peltier heating or cooling devices,heat sinks, thermistors, thermocouples, and the like. In some instances,the temperature control module (or “temperature controller”) may providefor a programmable temperature change at a specified, adjustable timeprior to performing specific assay or analysis steps. In some instances,the temperature controller may provide for programmable changes intemperature over specified time intervals. In some embodiments, thetemperature controller may further provide for cycling of temperaturesbetween two or more set temperatures with specified frequency and ramprates so that thermal cycling for amplification reactions may beperformed.

FIG. 34 illustrates one non-limiting example for temperature control ofthe flow cells (e.g., capillary flow cells or microfluidic device-basedflow cells) through the use of a metal plate that is placed in contactwith the flow cell cartridge. In some instances, the metal plate may beintegrated with the cartridge chassis. In some instances, the metalplate may be temperature controlled using a Peltier or resistive heater.

FIG. 35 illustrates one non-limiting approach for temperature control ofthe flow cells (e.g., capillary or microfluidic channel flow cells) thatcomprises a non-contact thermal control mechanism. In this approach, astream of temperature-controlled air is directed through the flow cellcartridge (e.g., towards a single capillary flow cell device or amicrofluidic channel flow cell device) using an air temperature controlsystem. The air temperature control system comprises a heat exchanger,e.g., a resistive heater coil, fins attached to a Peltier device, etc.,that is capable of heating and/or cooling the air and holding it at aconstant, user-specified temperature. The air temperature control systemalso comprises an air delivery device, such as a fan, that directs thestream of heated or cooled air to the capillary flow cell cartridge. Insome instances, the air temperature control system may be set to aconstant temperature T1 so that the air stream, and consequently theflow cell or cartridge (e.g., capillary flow cell or microfluidicchannel flow cell) is kept at a constant temperature T2, which in somecases may differ from the set temperature T1 depending on theenvironment temperature, air flow rate, etc. In some instances, two ormore such air temperature control systems may be installed around thecapillary flow cell device or flow cell cartridge so that the capillaryor cartridge may be rapidly cycled between several differenttemperatures by controlling which one of the air temperature controlsystems is active at a given time. In another approach, the temperaturesetting of the air temperature control system may be varied so thetemperature of the capillary flow cell or cartridge may be changedaccordingly.

Fluid dispensing robotics: In some implementations, the disclosedsystems may comprise an automated, programmable fluid-dispensing (orliquid-dispensing) system for use in dispensing reagents or othersolutions into, e.g., microplates, capillary flow cell devices andcartridges, microfluidic devices and cartridges, etc. Suitableautomated, programmable fluid-dispensing systems are commerciallyavailable from a number of vendors, e.g. Beckman Coulter, Perkin Elmer,Tecan, Velocity 11, and many others. In a preferred aspect of thedisclosed systems, the fluid-dispensing system further comprises amultichannel dispense head, e.g. a 4 channel, 8 channel, 16 channel, 96channel, or 384 channel dispense head, for simultaneous delivery ofprogrammable volumes of liquid (e.g. ranging from about 1 microliter toseveral milliliters) to multiple wells or locations on a flow cellcartridge or microfluidic cartridge.

Cartridge- and/or microplate-handling (pick-and-place) robotics: In someimplementations, the disclosed system may comprise a cartridge- and/ormicroplate-handling robotic system for automated replacement andpositioning of microplates, capillary flow cell cartridges, ormicrofluidic device cartridges in relation to the optical imagingsystem, or for optionally moving microplates, capillary flow cellcartridges, or microfluidic device cartridges between the opticalimaging system and a fluid-dispensing system. Suitable automated,programmable microplate-handling robotic systems are commerciallyavailable from a number of vendors, including Beckman Coulter, PerkinElemer, Tecan, Velocity 11, and many others. In a preferred aspect ofthe disclosed systems, an automated microplate-handling robotic systemis configured to move collections of microwell plates comprising samplesand/or reagents to and from, e.g., refrigerated storage units.

Spectroscopy or imaging modules: As indicated above, in someimplementations the disclosed analysis systems will include opticalimaging capabilities and may also include other spectroscopicmeasurement capabilities. For example, the disclosed imaging modules maybe configured to operate in any of a variety of imaging modes known tothose of skill in the art including, but not limited to, bright-field,dark-field, fluorescence, luminescence, or phosphorescence imaging. Insome instances, the one or more capillary flow cells or microfluidicdevices of a fluidics sub-system comprise a window that allows at leasta section of one or more capillaries or one or more fluid channels ineach flow cell or microfluidic device to be illuminated and imaged.

In some embodiments, single wavelength excitation and emissionfluorescence imaging may be performed. In some embodiments, dualwavelength excitation and emission (or multi-wavelength excitation oremission) fluorescence imaging may be performed. In some instances, theimaging module is configured to acquire video images. The choice ofimaging mode may impact the design of the flow cells devices orcartridges in that all or a portion of the capillaries or cartridge willnecessarily need to be optically transparent over the spectral range ofinterest. In some instances, a plurality of capillaries within acapillary flow cell cartridge may be imaged in their entirety within asingle image. In some instances, only a single capillary or a subset ofcapillaries within a capillary flow cell cartridge, or portions thereof,may be imaged within a single image. In some instances, a series ofimages may be “tiled” to create a single high-resolution image of one,two, several, or the entire plurality of capillaries within a cartridge.In some instances, a plurality of fluid channels within a microfluidicchip may be imaged in their entirety within a single image. In someinstances, only a single fluid channel or a subset of fluid channelswithin a microfluidic chip, or portions thereof, may be imaged within asingle image.

In some instances, a series of images may be “tiled” to create a singlehigh-resolution image of one, two, several, or the entire plurality offluid channels within a cartridge.

A spectroscopy or imaging module may comprise, e.g., a microscopeequipped with a CMOS of CCD camera. In some instances, the spectroscopyor imaging module may comprise, e.g., a custom instrument such as one ofthe imaging modules described herein that is configured to perform aspecific spectroscopic or imaging technique of interest. In general, thehardware associated with the spectroscopy or imaging module may includelight sources, detectors, and other optical components, as well asprocessors or computers.

Light sources: Any of a variety of light sources may be used to providethe imaging or excitation light, including but not limited to, tungstenlamps, tungsten-halogen lamps, arc lamps, lasers, light emitting diodes(LEDs), or laser diodes. In some instances, a combination of one or morelight sources, and additional optical components, e.g. lenses, filters,apertures, diaphragms, mirrors, and the like, may be configured as anillumination system (or sub-system).

Detectors: Any of a variety of image sensors may be used for imagingpurposes, including but not limited to, photodiode arrays,charge-coupled device (CCD) cameras, or complementarymetal-oxide-semiconductor (CMOS) image sensors. As used herein, “imagingsensors” may be one-dimensional (linear) or two-dimensional arraysensors. In many instances, a combination of one or more image sensors,and additional optical components, e.g. lenses, filters, apertures,diaphragms, mirrors, and the like, may be configured as an imagingsystem (or sub-system). In some instances, e.g., where spectroscopicmeasurements are performed by the system rather than imaging, suitabledetectors may include, but are not limited to, photodiodes, avalanchephotodiodes, and photomultipliers.

Other optical components: The hardware components of the spectroscopicmeasurement or imaging module may also include a variety of opticalcomponents for steering, shaping, filtering, or focusing light beamsthrough the system. Examples of suitable optical components include, butare not limited to, lenses, mirrors, prisms, apertures, diffractiongratings, colored glass filters, long-pass filters, short-pass filters,bandpass filters, narrowband interference filters, broadbandinterference filters, dichroic reflectors, optical fibers, opticalwaveguides, and the like. In some instances, as noted above, thespectroscopic measurement or imaging module may further comprise one ormore translation stages or other motion control mechanisms for thepurpose of moving capillary flow cell devices and cartridges relative tothe illumination and/or detection/imaging sub-systems, or vice versa.

Total internal reflection: In some instances, the optical module orsub-system may be designed to use all or a portion of an opticallytransparent wall of the capillaries or microfluidic channels in flowcell devices and cartridges as a waveguide for delivering excitationlight to the capillary or channel lumen(s) via total internalreflection. When incident excitation light strikes the surface of thecapillary or channel lumen at an angle with respect to a normal to thesurface that is larger than the critical angle (determined by therelative refractive indices of the capillary or channel wall materialand the aqueous buffer within the capillary or channel), total internalreflection occurs at the surface and the light propagates through thecapillary or channel wall along the length of the capillary or channel.Total internal reflection generates an evanescent wave at the lumensurface which penetrates the lumen interior for extremely shortdistances, and which may be used to selectively excite fluorophores atthe surface, e.g., labeled nucleotides that have been incorporated by apolymerase into a growing oligonucleotide through a solid-phase primerextension reaction.

Light-tight housings and environmental control chambers: In someimplementations, the disclosed systems may comprise a light-tighthousing to prevent stray ambient light from creating glare andobscuring, e.g., relatively faint fluorescence signals. In someimplementations, the disclosed systems may comprise an environmentalcontrol chamber that enables the system to operate under a tightlycontrolled temperature, humidity level, etc.

Processors and computers: In some instances, the disclosed systems maycomprise one or more processors or computers. The processor may be ahardware processor such as a central processing unit (CPU), a graphicprocessing unit (GPU), a general-purpose processing unit, or a computingplatform. The processor may be comprised of any of a variety of suitableintegrated circuits, microprocessors, logic devices, field-programmablegate arrays (FPGAs) and the like. In some instances, the processor maybe a single core or multi core processor, or a plurality of processorsmay be configured for parallel processing. Although the disclosure isdescribed with reference to a processor, other types of integratedcircuits and logic devices are also applicable. The processor may haveany suitable data operation capability. For example, the processor mayperform 512 bit, 256 bit, 128 bit, 64 bit, 32 bit, or 16 bit dataoperations.

The processor or CPU can execute a sequence of machine-readableinstructions, which can be embodied in a program or software. Theinstructions may be stored in a memory location. The instructions can bedirected to the CPU, which can subsequently program or otherwiseconfigure the CPU to implement, e.g., the system control methods of thepresent disclosure. Examples of operations performed by the CPU caninclude fetch, decode, execute, and write back.

Some processors may comprise a processing unit of a computer system. Thecomputer system may enable cloud-based data storage and/or computing. Insome instances, the computer system may be operatively coupled to acomputer network (“network”) with the aid of a communication interface.The network may be the internet, an intranet and/or extranet, anintranet and/or extranet that is in communication with the internet, ora local area network (LAN). The network in some cases is atelecommunication and/or data network. The network may include one ormore computer servers, which may enable distributed computing, such ascloud-based computing.

The computer system may also include computer memory or memory locations(e.g., random-access memory, read-only memory, flash memory), electronicstorage units (e.g., hard disk), communication interfaces (e.g., networkadapters) for communicating with one or more other systems, andperipheral devices, such as cache, other memory units, data storageunits and/or electronic display adapters. In some instances, thecommunication interface may allow the computer to be in communicationwith one or more additional devices. The computer may be able to receiveinput data from the coupled devices for analysis. Memory units, storageunits, communication interfaces, and peripheral devices may be incommunication with the processor or CPU through a communication bus(solid lines), such as may be incorporated into a motherboard. A memoryor storage unit may be a data storage unit (or data repository) forstoring data. The memory or storage units may store files, such asdrivers, libraries and saved programs. The memory or storage units maystore user data, e.g., user preferences and user programs.

The system control, image processing, and/or data analysis methods asdescribed herein can be implemented by way of machine-executable codestored in an electronic storage location of the computer system, suchas, for example, in the memory or electronic storage unit. Themachine-executable or machine-readable code can be provided in the formof software. During use, the code can be executed by the processor. Insome cases, the code can be retrieved from the storage unit and storedin memory for ready access by the processor. In some situations, theelectronic storage unit can be precluded, and machine-executableinstructions are stored in memory.

In some instances, the code may be pre-compiled and configured for usewith a machine having a processer adapted to execute the code. In someinstances, the code may be compiled during runtime. The code can besupplied in a programming language that can be selected to enable thecode to execute in a pre-compiled or as-compiled fashion.

Some aspects of the systems and methods provided herein can be embodiedin software. Various aspects of the technology may be thought of as“products” or “articles of manufacture” typically in the form of machine(or processor) executable code and/or associated data that is carried onor embodied in a type of machine-readable medium. Machine-executablecode can be stored on an electronic storage unit, such as memory (e.g.,read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory ofthe computers, processors or the like, or associated modules thereof,such as various semiconductor memories, tape drives, disk drives and thelike, which may provide non-transitory storage at any time for thesoftware programming. All or portions of the software may at times becommunicated through the Internet or various other telecommunicationnetworks. Such communications, for example, may enable loading of thesoftware from one computer or processor into another, for example, froma management server or host computer into the computer platform of anapplication server. Thus, another type of media that may bear thesoftware elements includes optical, electrical and electromagneticwaves, such as used across physical interfaces between local devices,through wired and optical landline networks and over various air-links.The physical elements that carry such waves, such as wired or wirelesslinks, optical links or the like, also may be considered as mediabearing the software. As used herein, unless restricted tonon-transitory, tangible “storage” media, terms such as computer ormachine “readable medium” refer to any medium that participates inproviding instructions to a processor for execution.

In some instances, the system control, image processing, and/or dataanalysis methods of the present disclosure may be implemented by way ofone or more algorithms. An algorithm may be implemented by way ofsoftware upon execution by the central processing unit.

System control software: In some instances, the system may comprise acomputer (or processor) and a computer-readable medium that includescode for providing a user interface as well as manual, semi-automated,or fully-automated control of all system functions, e.g., control of thefluid flow control module(s), the temperature control module(s), and/orthe spectroscopy or imaging module(s), as well as other data analysisand display options. The system computer or processor may be anintegrated component of the system (e.g. a microprocessor or motherboard embedded within the instrument) or may be a stand-alone module,for example, a main frame computer, a personal computer, or a laptopcomputer. Examples of fluid flow control functions provided by thesystem control software include, but are not limited to, volumetricfluid flow rates, fluid flow velocities, the timing and duration forsample and reagent addition, buffer addition, and rinse steps. Examplesof temperature control functions provided by the system control softwareinclude, but are not limited to, specifying temperature set point(s) andcontrol of the timing, duration, and ramp rates for temperature changes.Examples of spectroscopic measurement or imaging control functionsprovided by the system control software include, but are not limited to,autofocus capability, control of illumination or excitation lightexposure times and intensities, control of image acquisition rate,exposure time, and data storage options.

Image processing software: In some instances, the system may furthercomprise a computer (or processor) and computer-readable medium thatincludes code for providing image processing and analysis capability.Examples of image processing and analysis capability that may beprovided by the software include, but are not limited to, manual,semi-automated, or fully-automated image exposure adjustment (e.g. whitebalance, contrast adjustment, signal-averaging and other noise reductioncapability, etc.), automated edge detection and object identification(e.g., for identifying clonally-amplified clusters offluorescently-labeled oligonucleotides on the lumen surface of capillaryflow cell devices), automated statistical analysis (e.g., fordetermining the number of clonally-amplified clusters ofoligonucleotides identified per unit area of the capillary lumensurface, or for automated nucleotide base-calling in nucleic acidsequencing applications), and manual measurement capabilities (e.g. formeasuring distances between clusters or other objects, etc.).Optionally, instrument control and image processing/analysis softwaremay be written as separate software modules. In some embodiments,instrument control and image processing/analysis software may beincorporated into an integrated package.

Any of a variety of image processing methods known to those of skill inthe art may be used for image processing/pre-processing. Examplesinclude, but are not limited to, Canny edge detection methods,Canny-Deriche edge detection methods, first-order gradient edgedetection methods (e.g., the Sobel operator), second order differentialedge detection methods, phase congruency (phase coherence) edgedetection methods, other image segmentation algorithms (e.g., intensitythresholding, intensity clustering methods, intensity histogram-basedmethods, etc.), feature and pattern recognition algorithms (e.g., thegeneralized Hough transform for detecting arbitrary shapes, the circularHough transform, etc.), and mathematical analysis algorithms (e.g.,Fourier transform, fast Fourier transform, wavelet analysis,auto-correlation, etc.), or any combination thereof

Nucleic acid sequencing systems & applications: Nucleic acid sequencingprovides one non-limiting example of an application for the disclosedflow cell devices (e.g., capillary flow cell devices or cartridges andmicrofluidic devices and cartridges) and imaging systems. Many “secondgeneration” and “third generation” sequencing technologies utilize amassively parallel, cyclic array approach to performsequencing-by-nucleotide incorporation, in which accurate decoding of asingle-stranded template oligonucleotide sequence tethered to a solidsupport relies on successfully classifying signals that arise from thestepwise addition of A, G, C, and T nucleotides by a polymerase to acomplementary oligonucleotide strand. These methods typically requirethe oligonucleotide template to be modified with a known adaptersequence of fixed length, affixed to a solid support (e.g., the lumensurface(s) of the disclosed capillary flow cell devices or microfluidicchips) in a random or patterned array by hybridization tosurface-tethered capture probes (also referred to herein as “adapters”or “primers” tethered to the interior flow cell surfaces) of knownsequence that are complementary to that of the adapter sequence, andthen probed through a cyclic series of single base addition primerextension reactions that use, e.g., fluorescently-labeled nucleotides toidentify the sequence of bases in the template oligonucleotides. Theseprocesses thus require the use of miniaturized fluidics systems thatoffer precise, reproducible control of the timing of reagentintroduction to the flow cell in which the sequencing reactions areperformed, and small volumes to reduce or minimize the consumption ofcostly reagents.

Existing commercially-available NGS flow cells are constructed fromlayers of glass that have been etched, lapped, and/or processed by othermethods to meet the tight dimensional tolerances required for imaging,cooling, and/or other requirements. When flow cells are used asconsumables, the costly manufacturing processes required for theirfabrication result in costs per sequencing run that are too high to makesequencing routinely accessible to scientists and medical professionalsin the research and clinical fields.

This disclosure provides an example of a low-cost flow cell architecturethat includes low cost glass or polymer capillaries or microfluidicchannels, fluidics adapters, and cartridge chassis. Utilizing glass orpolymer capillaries that are extruded in their final cross-sectionalgeometry may eliminate the need for multiple high-precision and costlyglass manufacturing processes. Robustly constraining the orientation ofthe capillaries or microfluidic channels and providing convenientfluidic connections using molded plastic and/or elastomeric componentsfurther reduces cost. Laser bonding the components of the polymercartridge chassis provides a fast and efficient means of sealing thecapillary or the microfluidic channels and structurally stabilizing thecapillaries or channels and flow cell cartridge without requiring theuse of fasteners or adhesives.

The disclosed devices and systems may be configured to perform nucleicacid sequencing using any of a variety of “sequencing-by-nucleotideincorporation”, “sequencing-by-nucleotide binding”,“sequencing-by-nucleotide base-pairing”, and “sequencing-by-avidity”sequencing biochemistries. The improvements in flow cell device designdisclosed herein, e.g., comprising hydrophilic coated surfaces thatmaximize foreground signals for, e.g., fluorescently-labeled nucleicacid clusters disposed thereon, while minimizing background signal maygive rise to improvements in CNR for images used for base-callingpurposes, in combination with improvements in optical imaging systemdesign for fast dual-surface flow cell imaging (comprising simultaneousor near-simultaneous imaging of the interior flow cell surfaces)achieved through improved objective lens and/or tube lens designs thatprovide for larger depth of field and larger fields-of-view, and reducedreagent consumption (achieved through improved flow cell design) maygive rise to dramatic improvements in base-calling accuracy, shortenedimaging cycle times, shortened overall sequencing reaction cycle times,and higher throughput nucleic acid sequencing at reduced cost per base.

The systems disclosed herein may be configured to implement any of avariety of different sequencing methodologies using a variety ofdifferent sequencing chemistries. For example, FIG. 40 provides anon-limiting example of a flow chart for implementing asequencing-by-avidity method. A polymer-nucleotide conjugate may be usedto form a multivalent binding complex with a plurality of primed targetnucleic acid sequences tethered to a support surface, e.g., one or moreinterior surfaces of a flow cell, such that the multivalent bindingcomplex exhibits a significantly longer persistence time than affordedby the binding interactions between single nucleotides and single primedtarget nucleic acid sequences. In general, such a sequencing-by-avidityapproach will comprise one or more of the following steps: hybridizationof target nucleic acid sequences to adapter/primer sequences tethered tothe support surface; clonal amplification to create clusters ofamplified target sequences on the support surface; contacting thesupport surface with a polymer-nucleotide conjugate comprising aplurality of nucleotide moieties conjugated to a polymer core, whereinthe polymer-nucleotide conjugate may further comprise one or moredetectable labels, e.g., fluorophores, to create a stable, multivalentbinding complex; washing out of any excess, unbound polymer-nucleotideconjugate; detection of multivalent binding complexes, e.g., byfluorescence imaging of the support surface; identification of anucleotide in the target nucleic acid sequence (base-calling);destabilization of the multivalent binding complex, e.g., by changingthe ionic strength, ionic composition, and/or pH of the buffer; rinsingof the flow cell; and performing a primer extension reaction to add anucleotide comprising the complementary base for the nucleotide that wasidentified. The cycle may be repeated to identify additional nucleotidebases in the sequence, followed by processing and assembly of thesequence data. In some instances, data processing may comprisecalculation of sequencing performance metrics, such as a Q-score, inreal-time as the sequencing run is performed or as part of a post-rundata processing step.

In some instances, the disclosed hydrophilic, polymer coated flow celldevices used in combination with the optical imaging systems disclosedherein may confer one or more of the following additional advantages fora nucleic acid sequencing system: (i) decreased fluidic wash times (dueto reduced non-specific binding, and thus faster sequencing cycletimes), (ii) decreased imaging times (and thus faster turnaround timesfor assay readout and sequencing cycles), (iii) decreased overall workflow time requirements (due to decreased cycle times), (iv) decreaseddetection instrumentation costs (due to the improvements in CNR), (v)improved readout (base-calling) accuracy (due to improvements in CNR),(vi) improved reagent stability and decreased reagent usage requirements(and thus reduced reagents costs), and (vii) fewer run-time failures dueto nucleic acid amplification failures.

The methods, devices, and systems disclosed herein for performingnucleic acid sequencing are suitable for a variety of sequencingapplications and for sequencing nucleic acid molecules derived from anyof a variety of samples and sources. Nucleic acids, in some instances,may be extracted from any of a variety of biological samples, e.g.,blood samples, saliva samples, urine samples, cell samples, tissuesamples, and the like. For example, the disclosed devices and systemsmay be used for the analysis of nucleic acid molecules derived from anyof a variety of different cell, tissue, or sample types known to thoseof skill in the art. For example, nucleic acids may be extracted fromcells, or tissue samples comprising one or more types of cells, derivedfrom eukaryotes (such as animals, plants, fungi, protista),archaebacteria, or eubacteria. In some cases, nucleic acids may beextracted from prokaryotic or eukaryotic cells, such as adherent ornon-adherent eukaryotic cells. Nucleic acids are variously extractedfrom, for example, primary or immortalized rodent, porcine, feline,canine, bovine, equine, primate, or human cell lines. Nucleic acids maybe extracted from any of a variety of different cell, organ, or tissuetypes (e.g., white blood cells, red blood cells, platelets, epithelialcells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts,skeletal muscle cells, smooth muscle cells, gametes, or cells from theheart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder,stomach, colon, or small intestine). Nucleic acids may be extracted fromnormal or healthy cells. Alternately or in combination, acids areextracted from diseased cells, such as cancerous cells, or frompathogenic cells that are infecting a host. Some nucleic acids may beextracted from a distinct subset of cell types, e.g., immune cells (suchas T cells, cytotoxic (killer) T cells, helper T cells, alpha beta Tcells, gamma delta T cells, T cell progenitors, B cells, B-cellprogenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes,granulocytes, Natural Killer cells, plasma cells, memory cells,neutrophils, eosinophils, basophils, mast cells, monocytes, dendriticcells, and/or macrophages, or any combination thereof), undifferentiatedhuman stem cells, human stem cells that have been induced todifferentiate, rare cells (e.g., circulating tumor cells (CTCs),circulating epithelial cells, circulating endothelial cells, circulatingendometrial cells, bone marrow cells, progenitor cells, foam cells,mesenchymal cells, or trophoblasts). Other cells are contemplated andconsistent with the disclosure herein.

Nucleic acids may optionally be attached to one or more non-nucleotidemoieties such as labels and other small molecules, large molecules (suchas proteins, lipids, sugars, etc.), and solid or semi-solid supports,for example through covalent or non-covalent linkages with either the 5′or 3′ end of the nucleic acid. Labels include any moiety that isdetectable using any of a variety of detection methods known to those ofskill in the art, and thus renders the attached oligonucleotide ornucleic acid similarly detectable. Some labels, e.g., fluorophores, emitelectromagnetic radiation that is optically detectable or visible.Alternately or in combination, some labels comprise a mass tag thatrenders the labeled oligonucleotide or nucleic acid visible in massspectral data, or a redox tag that renders the labeled oligonucleotideor nucleic acid detectable by amperometry or voltametry. Some labelscomprise a magnetic tag that facilitates separation and/or purificationof the labeled oligonucleotide or nucleic acid. The nucleotide orpolynucleotide is often not attached to a label, and the presence of theoligonucleotide or nucleic acid is directly detected.

Flow cell devices configured for sequencing: In some instances, one ormore flow cell devices according to the present disclosure may beconfigured for nucleic acid sequencing applications, e.g., wherein twoor more interior flow cell device surfaces comprise hydrophilic polymercoatings that further comprise one or more capture oligonucleotides,e.g., adapter/primer oligonucleotides, or any other oligonucleotides asdisclosed herein. In some instances, the hydrophilic, polymer-coatedsurfaces of the disclosed flow cell devices may comprise a plurality ofoligonucleotides tethered thereto that have been selected for use insequencing a eukaryotic genome. In some instances, the hydrophilic,polymer-coated surfaces of the disclosed flow cell devices may comprisea plurality of oligonucleotides tethered thereto that have been selectedfor use in sequencing a prokaryotic genome or portion thereof. In someinstances, the hydrophilic, polymer-coated surfaces of the disclosedflow cell devices may comprise a plurality of oligonucleotides tetheredthereto that have been selected for use in sequencing a viral genome orportion thereof. In some instances, the hydrophilic, polymer-coatedsurfaces of the disclosed flow cell devices may comprise a plurality ofoligonucleotides tethered thereto that have been selected for use insequencing a transcriptome.

In some instances, a flow cell device of the present disclosure maycomprise a first surface in an orientation generally facing the interiorof the flow channel, a second surface in an orientation generally facingthe interior of the flow channel and further generally facing orparallel to the first surface, a third surface generally facing theinterior of a second flow channel, and a fourth surface, generallyfacing the interior of the second flow channel and generally opposed toor parallel to the third surface; wherein said second and third surfacesmay be located on or attached to opposite sides of a generally planarsubstrate which may be a reflective, transparent, or translucentsubstrate. In some instances, an imaging surface or imaging surfaceswithin a flow cell may be located within the center of a flow cell orwithin or as part of a division between two subunits or subdivisions ofa flow cell, wherein said flow cell may comprise a top surface and abottom surface, one or both of which may be transparent to suchdetection mode as may be utilized; and wherein a surface comprisingoligonucleotides adapters/primers tethered to one or more polymercoatings may be placed or interposed within the lumen of the flow cell.In some instances, the top and/or bottom surfaces do not includeattached oligonucleotide adapters/primers. In some instances, said topand/or bottom surfaces do comprise attached oligonucleotideadapters/primers. In some instances, either said top or said bottomsurface may comprise attached oligonucleotide adapters/primers. Asurface or surfaces placed or interposed within the lumen of a flow cellmay be located on or attached to one side, to an opposite side, or toboth sides of a generally planar substrate which may be a reflective,transparent, or translucent substrate.

In general, at least one layer of the one or more layers of lownonspecific binding coating on the flow cell device surfaces maycomprise functional groups for covalently or non-covalently attachingoligonucleotide molecules, e.g., adapter or primer sequences, or the atleast one layer may already comprise covalently or non-covalentlyattached oligonucleotide adapter or primer sequences at the time that itis deposited on the support surface. In some instances, theoligonucleotides tethered to the polymer molecules of at least one thirdlayer may be distributed at a plurality of depths throughout the layer.

In some instances, the oligonucleotide adapter or primer molecules arecovalently coupled to the polymer in solution, i.e., prior to couplingor depositing the polymer on the surface. In some instances, theoligonucleotide adapter or primer molecules are covalently coupled tothe polymer after it has been coupled to or deposited on the surface. Insome instances, at least one hydrophilic polymer layer comprises aplurality of covalently-attached oligonucleotide adapter or primermolecules. In some instances, at least two, at least three, at leastfour, or at least five layers of hydrophilic polymer comprise aplurality of covalently-attached adapter or primer molecules.

In some instances, the oligonucleotide adapter or primer molecules maybe coupled to the one or more layers of hydrophilic polymer using any ofa variety of suitable conjugation chemistries known to those of skill inthe art. For example, the oligonucleotide adapter or primer sequencesmay comprise moieties that are reactive with amine groups, carboxylgroups, thiol groups, and the like. Examples of suitable amine-reactiveconjugation chemistries that may be used include, but are not limitedto, reactions involving isothiocyanate, isocyanate, acyl azide, NHSester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,carbonate, aryl halide, imidoester, carbodiimide, anhydride, andfluorophenyl ester groups. Examples of suitable carboxyl-reactiveconjugation chemistries include, but are not limited to, reactionsinvolving carbodiimide compounds, e.g., water soluble EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCL). Examples ofsuitable sulfydryl-reactive conjugation chemistries include maleimides,haloacetyls and pyridyl disulfides.

One or more types of oligonucleotide molecules may be attached ortethered to the support surface. In some instances, the one or moretypes of oligonucleotide adapters or primers may comprise spacersequences, adapter sequences for hybridization to adapter-ligatedtemplate library nucleic acid sequences, forward amplification primers,reverse amplification primers, sequencing primers, and/or molecularbarcoding sequences, or any combination thereof. In some instances, 1primer or adapter sequence may be tethered to at least one layer of thesurface. In some instances, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 different primer or adapter sequences may be tethered to atleast one layer of the surface.

In some instances, the tethered oligonucleotide adapter and/or primersequences may range in length from about 10 nucleotides to about 100nucleotides. In some instances, the tethered oligonucleotide adapterand/or primer sequences may be at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, or at least 100 nucleotides in length. In some instances, thetethered oligonucleotide adapter and/or primer sequences may be at most100, at most 90, at most 80, at most 70, at most 60, at most 50, at most40, at most 30, at most 20, or at most 10 nucleotides in length. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the length of the tethered oligonucleotide adapter and/orprimer sequences may range from about 20 nucleotides to about 80nucleotides. Those of skill in the art will recognize that the length ofthe tethered oligonucleotide adapter and/or primer sequences may haveany value within this range, e.g., about 24 nucleotides.

In some instances, the number of coating layers and/or the materialcomposition of each layer is chosen so as to adjust the resultantsurface density of oligonucleotide adapters/primers (or other attachedmolecules) on the coated interior flow cell surfaces. In some instances,the surface density of oligonucleotide adapters/primers may range fromabout 1,000 primer molecules per μm² to about 1,000,000 primer moleculesper μm². In some instances, the surface density of oligonucleotideprimers may be at least 1,000, at least 10,000, at least 100,000, or atleast 1,000,000 molecules per μm². In some instances, the surfacedensity of oligonucleotide primers may be at most 1,000,000, at most100,000, at most 10,000, or at most 1,000 molecules per μm². Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the surface density of primers may range from about10,000 molecules per um² to about 100,000 molecules per um². Those ofskill in the art will recognize that the surface density of primermolecules may have any value within this range, e.g., about 455,000molecules per um². In some instances, the surface properties of thecapillary or channel lumen coating, including the surface density oftethered oligonucleotide primers, may be adjusted to improve oroptimize, e.g., solid-phase nucleic acid hybridization specificity andefficiency, and/or solid-phase nucleic acid amplification rate,specificity, and efficiency.

In some instances, the tethered adapter or primer sequences may comprisemodifications designed to facilitate the specificity and efficiency ofnucleic acid amplification as performed on the low-binding supports. Forexample, in some instances the primer may comprise polymerase stoppoints such that the stretch of primer sequence between the surfaceconjugation point and the modification site is always in single-strandedform and functions as a loading site for 5′ to 3′ helicases in somehelicase-dependent isothermal amplification methods. Other examples ofprimer modifications that may be used to create polymerase stop pointsinclude, but are not limited to, an insertion of a PEG chain into thebackbone of the primer between two nucleotides towards the 5′ end,insertion of an abasic nucleotide (i.e., a nucleotide that has neither apurine nor a pyrimidine base), or a lesion site which can be bypassed bythe helicase.

Nucleic acid hybridization: In some instances, the hydrophilic, polymercoated flow cell device surfaces disclosed herein may provide advantageswhen used alone or in combination with improved buffer formulations forperforming solid-phase nucleic acid hybridization and/or solid-phasenucleic acid amplification reactions as part of genotyping or nucleicacid sequencing applications. In some instances, the polymer-coated flowcell devices disclosed herein may provide advantages in terms ofimproved nucleic acid hybridization rate and specificity, and improvednucleic acid amplification rates and specificity that may be achievedthrough one or more of the following additional aspects of the presentdisclosure: (i) primer design (e.g., sequence and/or modifications),(ii) control of tethered primer density on the solid support, (iii) thesurface composition of the solid support, (iv) the surface polymerdensity of the solid support, (v) the use of improved hybridizationconditions before and during amplification, and/or (vi) the use ofimproved amplification formulations that decrease non-specific primeramplification or increase template amplification efficiency.

In some instances, it may be desirable to vary the surface density oftethered oligonucleotide adapters or primers on the coated flow cellsurfaces and/or the spacing of the tethered adapters or primers awayfrom the coated flow cell surface (e.g., by varying the length of alinker molecule used to tether the adapter or primers to the surface) inorder to “tune” the support for optimal performance when, e.g., using agiven amplification method. In some instances, adjusting the surfacedensity of tethered oligonucleotide adapters or primers may impact thelevel of specific and/or non-specific amplification observed on thesurface in a manner that varies according to the amplification methodselected. In some instances, the surface density of tetheredoligonucleotide adapters or primers may be varied by adjusting the ratioof molecular components used to create the support surface. For example,in the case that an oligonucleotide primer—PEG conjugate is used tocreate the final layer of a low-binding support, the ratio of theoligonucleotide primer—PEG conjugate to a non-conjugated PEG moleculemay be varied. The resulting surface density of tethered primermolecules may then be estimated or measured using any of a variety oftechniques known to those of skill in the art. Examples include, but arenot limited to, the use of radioisotope labeling and counting methods,covalent coupling of a cleavable molecule that comprises anoptically-detectable tag (e.g., a fluorescent tag) that may be cleavedfrom a support surface of defined area, collected in a fixed volume ofan appropriate solvent, and then quantified by comparison offluorescence signals to that for a calibration solution of known opticaltag concentration, or using fluorescence imaging techniques providedthat care has been taken with the labeling reaction conditions and imageacquisition settings to ensure that the fluorescence signals arelinearly related to the number of fluorophores on the surface (e.g.,that there is no significant self-quenching of the fluorophores on thesurface).

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, either alone or in combination with improved oroptimized buffer formulations, may yield relative hybridization ratesthat range from about 2× to about 20× faster than that for aconventional hybridization protocol. In some instances, the relativehybridization rate may be at least 2×, at least 3×, at least 4×, atleast 5×, at least 6×, at least 7×, at least 8×, at least 9×, at least10×, at least 12×, at least 14×, at least 16×, at least 18×, at least20×, at least 25×, at least 30×, or at least 40× that for a conventionalhybridization protocol.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, either alone or in combination with improved oroptimized buffer formulations, may yield total hybridization reactiontimes (i.e., the time required to reach 90%, 95%, 98%, or 99% completionof the hybridization reaction) of less than 60 minutes, 50 minutes, 40minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutesfor any of these completion metrics.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, either alone or in combination with improved oroptimized buffer formulations, may yield improved hybridizationspecificity compared to that for a conventional hybridization protocol.In some instances, the hybridization specificity that may be achieved isbetter than 1 base mismatch in 10 hybridization events, 1 base mismatchin20 hybridization events, 1 base mismatch in 30 hybridization events, 1base mismatch in 40 hybridization events, 1 base mismatch in 50hybridization events, 1 base mismatch in 75 hybridization events, 1 basemismatch in 100 hybridization events, 1 base mismatch in 200hybridization events, 1 base mismatch in 300 hybridization events, 1base mismatch in 400 hybridization events, 1 base mismatch in 500hybridization events, 1 base mismatch in 600 hybridization events, 1base mismatch in 700 hybridization events, 1 base mismatch in 800hybridization events, 1 base mismatch in 900 hybridization events, 1base mismatch in 1,000 hybridization events, 1 base mismatch in 2,000hybridization events, 1 base mismatch in 3,000 hybridization events, 1base mismatch in 4,000 hybridization events, 1 base mismatch in 5,000hybridization events, 1 base mismatch in 6,000 hybridization events, 1base mismatch in 7,000 hybridization events, 1 base mismatch in 8,000hybridization events, 1 base mismatch in 9,000 hybridization events, or1 base mismatch in 10,000 hybridization events.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, either alone or in combination with improved oroptimized buffer formulations, may yield improved hybridizationefficiency (e.g., the fraction of available oligonucleotide primers onthe support surface that are successfully hybridized with targetoligonucleotide sequences) compared to that for a conventionalhybridization protocol. In some instances, the hybridization efficiencythat may be achieved is better than 50%, 60%, 70%, 80%, 85%, 90%, 95%,98%, or 99% for any of the input target oligonucleotide concentrationsspecified below and in any of the hybridization reaction times specifiedabove. In some instances, e.g., wherein the hybridization efficiency isless than 100%, the resulting surface density of target nucleic acidsequences hybridized to the support surface may be less than the surfacedensity of oligonucleotide adapter or primer sequences on the surface.

In some instances, use of the disclosed hydrophilic, polymer-coated flowcell devices for nucleic acid hybridization (or nucleic acidamplification) applications using conventional hybridization (oramplification) protocols, or improved or optimized hybridization (oramplification) protocols, may lead to a reduced requirement for theinput concentration of target (or sample) nucleic acid moleculescontacted with the support surface. For example, in some instances, thetarget (or sample) nucleic acid molecules may be contacted with thesupport surface at a concentration ranging from about 10 pM to about 1μM(i.e., prior to annealing or amplification). In some instances, thetarget (or sample) nucleic acid molecules may be administered at aconcentration of at least 10 pM, at least 20 pM, at least 30 pM, atleast 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700pM, at least 800pM, at least 900 pM, at least 1 nM, at least 10 nM, atleast 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, atleast 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, atleast 600 nM, at leasy 700 nM, at least 800 nM, at least 900 nM, or atleast 1μM. In some instances, the target (or sample) nucleic acidmolecules may be administered at a concentration of at most 1μM, at most900 nM, at most 800 nm, at most 700 nM, at most 600 nM, at most 500 nM,at most 400 nM, at most 300 nM, at most 200 nM, at most 100 nM, at most90 nM, at most 80 nM, at most 70 nM, at most 60 nM, at most 50 nM, atmost 40 nM, at most 30 nM, at most 20 nM, at most 10 nM, at most 1 nM,at most 900 pM, at most 800 pM, at most 700 pM, at most 600 pM, at most500 pM, at most 400 pM, at most 300 pM, at most 200 pM, at most 100 pM,at most 90 pM, at most 80 pM, at most 70 pM, at most 60 pM, at most 50pM, at most 40 pM, at most 30 pM, at most 20 pM, or at most 10 pM. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the target (or sample) nucleic acid moleculesmay be administered at a concentration ranging from about 90 pM to about200 nM. Those of skill in the art will recognize that the target (orsample) nucleic acid molecules may be administered at a concentrationhaving any value within this range, e.g., about 855 nM.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, either alone or in combination with improved oroptimized hybridization buffer formulations, may result in a surfacedensity of hybridized target (or sample) oligonucleotide molecules(i.e., prior to performing any subsequent solid-phase or clonalamplification reaction) ranging from about from about 0.0001 targetoligonucleotide molecules per μm² to about 1,000,000 targetoligonucleotide molecules per μm². In some instances, the surfacedensity of hybridized target oligonucleotide molecules may be at least0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01,at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 200,at least 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900, at least 1,000, at least 1,500, at least 2,000,at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500,at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least9,000, at least 9,500, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, or at least 1,000,000 molecules per μm². Insome instances, the surface density of hybridized target oligonucleotidemolecules may be at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, atmost 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000,at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most800, at most 700, at most 600, at most 500, at most 400, at most 300, atmost 200, at most 100, at most 90, at most 80, at most 70, at most 60,at most 50, at most 40, at most 30, at most 20, at most 10, at most 5,at most 1, at most 0.5, at most 0.1, at most 0.05, at most 0.01, at most0.005, at most 0.001, at most 0.0005, or at most 0.0001 molecules perμm². Any of the lower and upper values described in this paragraph maybe combined to form a range included within the present disclosure, forexample, in some instances the surface density of hybridized targetoligonucleotide molecules may range from about 3,000 molecules per μm²to about 20,000 molecules per μm². Those of skill in the art willrecognize that the surface density of hybridized target oligonucleotidemolecules may have any value within this range, e.g., about 2,700molecules per μm2.

Stated differently, in some instances the use of the disclosedlow-binding supports alone or in combination with improved or optimizedhybridization buffer formulations may result in a surface density ofhybridized target (or sample) oligonucleotide molecules (i.e., prior toperforming any subsequent solid-phase or clonal amplification reaction)ranging from about 100 hybridized target oligonucleotide molecules permm² to about 1×10¹² hybridized target oligonucleotide molecules per mm².In some instances, the surface density of hybridized targetoligonucleotide molecules may be at least 100, at least 500, at least1,000, at least 4,000, at least 5,000, at least 6,000, at least 10,000,at least 15,000, at least 20,000, at least 25,000, at least 30,000, atleast 35,000, at least 40,000, at least 45,000, at least 50,000, atleast 55,000, at least 60,000, at least 65,000, at least 70,000, atleast 75,000, at least 80,000, at least 85,000, at least 90,000, atleast 95,000, at least 100,000, at least 150,000, at least 200,000, atleast 250,000, at least 300,000, at least 350,000, at least 400,000, atleast 450,000, at least 500,000, at least 550,000, at least 600,000, atleast 650,000, at least 700,000, at least 750,000, at least 800,000, atleast 850,000, at least 900,000, at least 950,000, at least 1,000,000,at least 5,000,000, at least 1×10⁷, at least 5×10⁷, at least 1×10⁸, atleast 5×10⁸,at least 1×10⁹, at least 5×10⁹, at least 1 x 10¹⁰, at least5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least 1×10¹² moleculesper mm². In some instances, the surface density of hybridized targetoligonucleotide molecules may be at most 1×10¹², at most 5×10¹¹, at most1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most 1×10⁹, atmost 5×10⁸, at most 1×10⁸, at most 5×10⁷, at most 1×10⁷, at most5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm². Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of hybridized targetoligonucleotide molecules may range from about 5,000 molecules per mm²to about 50,000 molecules per mm². Those of skill in the art willrecognize that the surface density of hybridized target oligonucleotidemolecules may have any value within this range, e.g., about 50,700molecules per mm².

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) hybridized to the oligonucleotide adapter orprimer molecules attached to the low-binding support surface may rangein length from about 0.02 kilobases (kb) to about 20 kb or from about0.1 kilobases (kb) to about 20 kb. In some instances, the targetoligonucleotide molecules may be at least 0.001 kb, at least 0.005 kb,at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb inlength, at least 0.2 kb in length, at least 0.3 kb in length, at least0.4 kb in length, at least 0.5 kb in length, at least 0.6 kb in length,at least 0.7 kb in length, at least 0.8 kb in length, at least 0.9 kb inlength, at least 1 kb in length, at least 2 kb in length, at least 3 kbin length, at least 4 kb in length, at least 5 kb in length, at least 6kb in length, at least 7 kb in length, at least 8 kb in length, at least9 kb in length, at least 10 kb in length, at least 15 kb in length, atleast 20 kb in length, at least 30 kb in length, or at least 40 kb inlength, or any intermediate value spanned by the range described herein,e.g., at least 0.85 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-stranded,multimeric nucleic acid molecules (e.g., concatamers) further comprisingrepeats of a regularly occurring monomer unit. In some instances, thesingle-stranded or double-stranded, multimeric nucleic acid moleculesmay be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least0.02 kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb inlength, at least 0.3 kb in length, at least 0.4 kb in length, at least0.5 kb in length, at least 1 kb in length, at least 2 kb in length, atleast 3 kb in length, at least 4 kb in length, at least 5 kb in length,at least 6 kb in length, at least 7 kb in length, at least 8 kb inlength, at least 9 kb in length, at least 10 kb in length, at least 15kb in length, or at least 20 kb in length, at least 30 kb in length, orat least 40 kb in length, or any intermediate value spanned by the rangedescribed herein, e.g., about 2.45 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-strandedmultimeric nucleic acid molecules (e.g., concatamers) comprising fromabout 2 to about 100 copies of a regularly repeating monomer unit. Insome instances, the number of copies of the regularly repeating monomerunit may be at least 2, at least 3, at least 4, at least 5, at least 10,at least 15, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 55, at least 60, at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, and at least 100. In some instances, the number of copies ofthe regularly repeating monomer unit may be at most 100, at most 95, atmost 90, at most 85, at most 80, at most 75, at most 70, at most 65, atmost 60, at most 55, at most 50, at most 45, at most 40, at most 35, atmost 30, at most 25, at most 20, at most 15, at most 10, at most 5, atmost 4, at most 3, or at most 2. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances the numberof copies of the regularly repeating monomer unit may range from about 4to about 60. Those of skill in the art will recognize that the number ofcopies of the regularly repeating monomer unit may have any value withinthis range, e.g., about 17. Thus, in some instances, the surface densityof hybridized target sequences in terms of the number of copies of atarget sequence per unit area of the support surface may exceed thesurface density of oligonucleotide primers even if the hybridizationefficiency is less than 100%.

Nucleic acid surface amplification (NASA): As used herein, the phrase“nucleic acid surface amplification” (NASA) is used interchangeably withthe phrase “solid-phase nucleic acid amplification” (or simply“solid-phase amplification”). In some aspects of the present disclosure,nucleic acid amplification formulations are described which, incombination with the disclosed hydrophilic, polymer-coated flow celldevices, provide for improved amplification rates, amplificationspecificity, and amplification efficiency. As used herein, specificamplification refers to amplification of template libraryoligonucleotide strands that have been tethered to the solid supporteither covalently or non-covalently. As used herein, non-specificamplification refers to amplification of primer-dimers or othernon-template nucleic acids. As used herein, amplification efficiency isa measure of the percentage of tethered oligonucleotides on the supportsurface that are successfully amplified during a given amplificationcycle or amplification reaction. Nucleic acid amplification performed onsurfaces disclosed herein may obtain amplification efficiencies of atleast 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%, such as 98% or99%.

Any of a variety of thermal cycling or isothermal nucleic acidamplification schemes may be used with the disclosed low-bindingsupports. Examples of nucleic acid amplification methods that may beutilized with the disclosed low-binding supports include, but are notlimited to, polymerase chain reaction (PCR), multiple displacementamplification (MDA), transcription-mediated amplification (TMA), nucleicacid sequence-based amplification (NASBA), strand displacementamplification (SDA), real-time SDA, bridge amplification, isothermalbridge amplification, rolling circle amplification, circle-to-circleamplification, helicase-dependent amplification, recombinase-dependentamplification, or single-stranded binding (SSB) protein-dependentamplification.

In some instances, improvements in amplification rate, amplificationspecificity, and amplification efficiency may be achieved using thedisclosed hydrophilic, polymer-coated flow cell devices, either alone orin combination with formulations of the amplification reactioncomponents. In addition to inclusion of nucleotides, one or morepolymerases, helicases, single-stranded binding proteins, etc. (or anycombination thereof), the amplification reaction mixture may be adjustedin a variety of ways to achieve improved performance including, but arenot limited to, choice of buffer type, buffer pH, organic solventmixtures, buffer viscosity, detergents and zwitterionic components,ionic strength (including adjustment of both monovalent and divalent ionconcentrations), antioxidants and reducing agents, carbohydrates, BSA,polyethylene glycol, dextran sulfate, betaine, other additives, and thelike.

The use of the disclosed hydrophilic, polymer-coated flow cell devices,alone or in combination with improved or optimized amplificationreaction formulations, may yield increased amplification rates comparedto those obtained using conventional supports and amplificationprotocols. In some instances, the relative amplification rates that maybe achieved may be at least 2×, at least 3×, at least 4×, at least 5×,at least 6×, at least 7×, at least 8×, at least 9×, at least 10×, atleast 12×, at least 14×, at least 16×, at least 18×, or at least 20xthat for use of conventional supports and amplification protocols forany of the amplification methods described above.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, alone or in combination with improved or optimizedbuffer formulations, may yield total amplification reaction times (i.e.,the time required to reach 90%, 95%, 98%, or 99% completion of theamplification reaction) of less than 180 mins, 120 mins, 90 min, 60minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10minutes, 5 minutes, 3 minutes, 1 minute, 50 s, 40s, 30s, 20s, or lOs forany of these completion metrics.

In some instances, the use of the disclosed low-binding supports aloneor in combination with improved or optimized amplification bufferformulations may enable faster amplification reaction times (i.e., thetimes required to reach 90%, 95%, 98%, or 99% completion of theamplification reaction) of no more than 60 minutes, 50 minutes, 40minutes, 30 minutes, 20 minutes, or 10 minutes. Similarly, use of thedisclosed low-binding supports alone or in combination with improved oroptimized buffer formulations may enable amplification reactions to becompleted in some cases in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or no more than 30 cycles.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, alone or in combination with improved or optimizedamplification reaction formulations, may yield increased specificamplification and/or decreased non-specific amplification compared tothat obtained using conventional supports and amplification protocols.In some instances, the resulting ratio of specificamplification-to-non-specific amplification that may be achieved is atleast 4:1 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1,70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1,800:1, 900:1, or 1,000:1.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, alone or in combination with improved or optimizedamplification reaction formulations, may yield increased amplificationefficiency compared to that obtained using conventional supports andamplification protocols. In some instances, the amplification efficiencythat may be achieved is better than 50%, 60%, 70% 80%, 85%, 90%, 95%,98%, or 99% in any of the amplification reaction times specified above.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) hybridized to theoligonucleotide adapter or primer molecules attached to the hydrophilic,polymer-coated flow cell device surfaces may range in length from about0.02 kilobases (kb) to about 20 kb or from about 0.1 kilobases (kb) toabout 20 kb. In some instances, the clonally-amplified targetoligonucleotide molecules may be at least 0.001 kb, at least 0.005 kb,at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb inlength, at least 0.2 kb in length, at least 0.3 kb in length, at least0.4 kb in length, at least 0.5 kb in length, at least 1 kb in length, atleast 2 kb in length, at least 3 kb in length, at least 4 kb in length,at least 5 kb in length, at least 6 kb in length, at least 7 kb inlength, at least 8 kb in length, at least 9 kb in length, at least 10 kbin length, at least 15 kb in length, or at least 20 kb in length, or anyintermediate value spanned by the range described herein, e.g., at least0.85 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded, multimeric nucleic acid molecules(e.g., concatamers) further comprising repeats of a regularly occurringmonomer unit. In some instances, the clonally-amplified single-strandedor double-stranded, multimeric nucleic acid molecules may be at least0.1 kb in length, at least 0.2 kb in length, at least 0.3 kb in length,at least 0.4 kb in length, at least 0.5 kb in length, at least 1 kb inlength, at least 2 kb in length, at least 3 kb in length, at least 4 kbin length, at least 5 kb in length, at least 6 kb in length, at least 7kb in length, at least 8 kb in length, at least 9 kb in length, at least10 kb in length, at least 15 kb in length, or at least 20 kb in length,or any intermediate value spanned by the range described herein, e.g.,about 2.45 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded multimeric nucleic acid (e.g.,concatamers) molecules comprising from about 2 to about 100 copies of aregularly repeating monomer unit. In some instances, the number ofcopies of the regularly repeating monomer unit may be at least 2, atleast 3, at least 4, at least 5, at least 10, at least 15, at least 20,at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, and at least100. In some instances, the number of copies of the regularly repeatingmonomer unit may be at most 100, at most 95, at most 90, at most 85, atmost 80, at most 75, at most 70, at most 65, at most 60, at most 55, atmost 50, at most 45, at most 40, at most 35, at most 30, at most 25, atmost 20, at most 15, at most 10, at most 5, at most 4, at most 3, or atmost 2. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the number of copies of the regularlyrepeating monomer unit may range from about 4 to about 60. Those ofskill in the art will recognize that the number of copies of theregularly repeating monomer unit may have any value within this range,e.g., about 12. Thus, in some instances, the surface density ofclonally-amplified target sequences in terms of the number of copies ofa target sequence per unit area of the support surface may exceed thesurface density of oligonucleotide primers even if the hybridizationand/or amplification efficiencies are less than 100%.

In some instances, the use of the disclosed hydrophilic, polymer-coatedflow cell devices, alone or in combination with improved or optimizedamplification reaction formulations, may yield increased clonal copynumber compared to that obtained using conventional supports andamplification protocols. In some instances, e.g., wherein theclonally-amplified target (or sample) oligonucleotide molecules compriseconcatenated, multimeric repeats of a monomeric target sequence, theclonal copy number may be substantially smaller than compared to thatobtained using conventional supports and amplification protocols. Thus,in some instances, the clonal copy number may range from about 1molecule to about 100,000 molecules (e.g., target sequence molecules)per amplified colony. In some instances, the clonal copy number may beat least 1, at least 5, at least 10, at least 50, at least 100, at least500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, atleast 5,000, at least 6,000, at least 7,000, at least 8,000, at least9,000, at least 10,000, at least 15,000, at least 20,000, at least25,000, at least 30,000, at least 35,000, at least 40,000, at least45,000, at least 50,000, at least 55,000, at least 60,000, at least65,000, at least 70,000, at least 75,000, at least 80,000, at least85,000, at least 90,000, at least 95,000, or at least 100,000 moleculesper amplified colony. In some instances, the clonal copy number may beat most 100,000, at most 95,000, at most 90,000, at most 85,000, at most80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000,at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000,at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, atmost 1,000, at most 500, at most 100, at most 50, at most 10, at most 5,or at most 1 molecule per amplified colony. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe clonal copy number may range from about 2,000 molecules to about9,000 molecules. Those of skill in the art will recognize that theclonal copy number may have any value within this range, e.g., about2,220 molecules in some instances, or about 2 molecules in others.

As noted above, in some instances the amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may compriseconcatenated, multimeric repeats of a monomeric target sequence. In someinstances, the amplified target (or sample) oligonucleotide molecules(or nucleic acid molecules) may comprise a plurality of molecules eachof which comprises a single monomeric target sequence. Thus, the use ofthe disclosed hydrophilic, polymer-coated flow cell devices, alone or incombination with improved or optimized amplification reactionformulations, may result in a surface density of target sequence copiesthat ranges from about 100 target sequence copies per mm² to about1×10¹² target sequence copies per mm². In some instances, the surfacedensity of target sequence copies may be at least 100, at least 500, atleast 1,000, at least 5,000, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, at least 1,000,000, at least 5,000,000, atleast 1×10⁷, at least 5×10⁷, at least 1×10⁸, at least 5×10⁸,at least1×10⁹, at least 5×10⁹, at least 1×10¹⁰, at least 5×10¹⁰, at least1×10¹¹, at least 5×10¹¹, or at least 1 x 10¹² of clonally amplifiedtarget sequence molecules per mm². In some instances, the surfacedensity of target sequence copies may be at most 1×10¹², at most 5×10¹¹,at most 1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most1×10⁹, at most 5×10⁸, at most 1×10⁸, at most 5×10⁷, at most 1×10⁷, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 target sequence copiesper mm². Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the surface density of target sequencecopies may range from about 1,000 target sequence copies per mm² toabout 65,000 target sequence copies mm². Those of skill in the art willrecognize that the surface density of target sequence copies may haveany value within this range, e.g., about 49,600 target sequence copiesper mm².

In some instances, the use of the disclosed low-binding supports aloneor in combination with improved or optimized amplification bufferformulations may result in a surface density of clonally-amplifiedtarget (or sample) oligonucleotide molecules (or clusters) ranging fromabout from about 100 molecules per mm2 to about 1×10¹² colonies per mm².In some instances, the surface density of clonally-amplified moleculesmay be at least 100, at least 500, at least 1,000, at least 5,000, atleast 10,000, at least 15,000, at least 20,000, at least 25,000, atleast 30,000, at least 35,000, at least 40,000, at least 45,000, atleast 50,000, at least 55,000, at least 60,000, at least 65,000, atleast 70,000, at least 75,000, at least 80,000, at least 85,000, atleast 90,000, at least 95,000, at least 100,000, at least 150,000, atleast 200,000, at least 250,000, at least 300,000, at least 350,000, atleast 400,000, at least 450,000, at least 500,000, at least 550,000, atleast 600,000, at least 650,000, at least 700,000, at least 750,000, atleast 800,000, at least 850,000, at least 900,000, at least 950,000, atleast 1,000,000, at least 5,000,000, at least 1×10⁷, at least 5×10⁷, atleast 1×10⁸, at least 5×10⁸,at least 1×10⁹, at least 5×10⁹, at least1×10¹⁰, at least 5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least1×10¹² molecules per mm². In some instances, the surface density ofclonally-amplified molecules may be at most 1×10¹², at most 5×10¹¹, atmost 1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most 1 x10⁹, at most 5×10⁸, at most 1×10⁸, at most 5×10⁷, at most 1×10⁷, at most5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm². Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedmolecules may range from about 5,000 molecules per mm² to about 50,000molecules per mm². Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 molecules per mm².

In some instances. the use of the disclosed hydrophilic, polymer-coatedflow cell devices, alone or in combination with improved or optimizedamplification reaction formulations, may yield signal from the amplifiedand labeled nucleic acid populations (e.g., a fluorescence signal) thathas a coefficient of variance of no greater than 50%, such as 50%, 40%,30%, 20%, 15%, 10%, 5%, or less than 5%.

Similarly, in some instances the use of the disclosed hydrophilic,polymer-coated flow cell devices, alone or in combination with improvedor optimized amplification reaction formulations, may yield signal fromthe amplified and non-labeled nucleic acid populations that has acoefficient of variance of no greater than 50%, such as 50%, 40%, 30%,20%, 10%, 5%, or less than 5%.

Fluorescence imaging of hydrophilic, polymer-coated flow cell devicesurfaces: The disclosed hydrophilic, polymer-coated flow cell devicescomprising, e.g., clonal clusters of labeled target nucleic acidmolecules disposed thereon may be used in any of a variety of nucleicacid analysis applications, e.g., nucleic acid base discrimination,nucleic acid base classification, nucleic acid base calling, nucleicacid detection applications, nucleic acid sequencing applications, andnucleic acid-based (genetic and genomic) diagnostic applications. Inmany of these applications, fluorescence imaging techniques may be usedto monitor hybridization, amplification, and/or sequencing reactionsperformed on the low-binding supports. Fluorescence imaging may beperformed using any of the optical imaging modules disclosed herein, aswell as a variety of fluorophores, fluorescence imaging techniques, andother fluorescence imaging instruments known to those of skill in theart.

In some instances, the performance of nucleic acid hybridization and/oramplification reactions using the disclosed hydrophilic, polymer-coatedflow cell devices and reaction buffer formulations may be assessed usingfluorescence imaging techniques, where the contrast-to-noise ratio (CNR)of the images provides a key metric in assessing amplificationspecificity and non-specific binding on the support. CNR is commonlydefined as: CNR =(Signal - Background)/Noise. The background term iscommonly taken to be the signal measured for the interstitial regionssurrounding a particular feature (diffraction limited spot, DLS) in aspecified region of interest (ROI). As noted above, whilesignal-to-noise ratio (SNR) is often considered to be a benchmark ofoverall signal quality, it can be shown that improved CNR can provide asignificant advantage over SNR as a benchmark for signal quality inapplications that require rapid image capture (e.g., sequencingapplications for which cycle times should be reduced or minimized). Athigh CNR, the imaging time required to reach accurate signaldiscrimination (and thus accurate base-calling in the case of sequencingapplications) can be drastically reduced even with moderate improvementsin CNR.

In most ensemble-based sequencing approaches, the background term istypically measured as the signal associated with ‘interstitial’ regions.In addition to “interstitial” background (Binter), “intrastitial”background (Bintra) exists within the discrete regions occupied byamplified DNA colonies. The combination of these two background signalterms dictates the achievable CNR in the image, and subsequentlydirectly impacts the optical instrument requirements, architecturecosts, reagent costs, run-times, cost/genome, and ultimately theaccuracy and data quality for cyclic array-based sequencingapplications. The Binter background signal arises from a variety ofsources; a few examples include auto-fluorescence from consumable flowcells, non-specific adsorption of detection molecules that yieldspurious fluorescence signals that may obscure the foreground signalfrom the ROI, and the presence of non-specific DNA amplificationproducts (e.g., those arising from primer dimers). In typical nextgeneration sequencing (NGS) applications, this background signal in thecurrent field-of-view (FOV) is averaged over time and subtracted. Thesignal arising from individual DNA colonies (i.e., (S)-Binter in theFOV) yields a discernable feature that can be classified. In someinstances, the intrastitial background (Bintra) can contribute aconfounding fluorescence signal that is not specific to the target ofinterest but is present in the same ROI, thus making it far moredifficult to average and subtract.

The implementation of nucleic acid amplification on the hydrophilic,polymer-coated substrate surfaces of the present disclosure may decreasethe Binter background signal by reducing non-specific binding, may leadto improvements in specific nucleic acid amplification, and may lead toa decrease in non-specific amplification that can impact the backgroundsignal arising from both the interstitial and intrastitial regions. Insome instances, the disclosed low nonspecific binding support surfaces,optionally used in combination with improved hybridization and/oramplification reaction buffer formulations, may lead to improvements inCNR by a factor of 2, 5, 10, 100, 200, 500, or 1000-fold over thoseachieved using conventional supports and hybridization, amplification,and/or sequencing protocols. Although described here in the context ofusing fluorescence imaging as the read-out or detection mode, the sameprinciples apply to the use of the disclosed low nonspecific bindingsupports and nucleic acid hybridization and amplification formulationsfor other detection modes as well, including both optical andnon-optical detection modes.

Alternative sequencing biochemistries: In addition to thesequencing-by-nucleotide incorporation approach described above, thedisclosed flow cell devices and optical imaging systems are compatiblewith other emerging nucleic acid sequencing biochemistries as well.Examples include the “sequencing-by-nucleotide binding” approachdescribed in U.S. Pat. No. 10,655,176 B2, and the“sequencing-by-avidity” approach described in U.S. Pat. No. 10,768,173B2.

The “sequencing-by-nucleotide binding” approach, as currently beingdeveloped by Omniome, Inc. (San Diego, Calif.) is based on performingrepetitive cycles of detecting a stabilized complex that forms at eachposition along the template (e.g. a ternary complex that includes theprimed template (tethered to a sample support structure), a polymerase,and a cognate nucleotide for the position), under conditions thatprevent covalent incorporation of the cognate nucleotide into theprimer, and then extending the primer to allow detection of the nextposition along the template. In the sequencing-by-binding approach,detection of the nucleotide at each position of the template occursprior to extension of the primer to the next position. Generally, themethodology is used to distinguish the four different nucleotide typesthat can be present at positions along a nucleic acid template byuniquely labelling each type of ternary complex (i.e. different types ofternary complexes differing in the type of nucleotide it contains) or byseparately delivering the reagents needed to form each type of ternarycomplex. In some instances, the labeling may comprise fluorescencelabeling of, e.g., the cognate nucleotide or the polymerase thatparticipate in the ternary complex. The approach is thus compatible withthe disclosed flow cell devices and imaging systems.

The “sequencing-by-avidity” approach, as currently being developed byElement Biosciences, Inc. (San Diego, CA) relies on the increasedavidity (or “functional affinity”) derived from forming a complexcomprising a plurality of individual non-covalent binding interactions.Element's approach is based on the detection of a multivalent bindingcomplex formed between a fluorescently-labeled polymer-nucleotideconjugate, a polymerase, and a plurality of primed target nucleic acidmolecules tethered to a sample support structure, which allows thedetection/base-calling step to be separated from the nucleotideincorporation step. Fluorescence imaging is used to detect the boundcomplex and thereby determine the identity of the N+1 nucleotide in thetarget nucleic acid sequence (where the primer extension strand is Nnucleotides in length). Following the imaging step, the multivalentbinding complex is disrupted and washed away, the correct blockednucleotide is incorporated into the primer extension strand, and thecycle is repeated.

In some instances, a polymer-nucleotide conjugate of the presentdisclosure may comprise a plurality of nucleotide moieties or nucleotideanalog moieties conjugated to a polymer core, e.g., through the 5′ endof the nucleotide, either directly or via a linker. By way ofnon-limiting example, the nucleotide moieties may include ribonucleotidemoieties, ribonucleotide analog moieties, deoxyribonucleotide moieties,deoxyribonucleotide analog moieties, or any combination thereof. In someinstances, the nucleotides or nucleotide analogs may comprisedeoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine,adenosine, guanosine, 5-methyl-uridine, and/or cytidine. In someinstances, the nucleotide or nucleotide analog moieties may comprise anucleotide that has been modified to inhibit elongation during apolymerase reaction or a sequencing reaction, such as wherein the atleast one nucleotide or nucleotide analog is a nucleotide that lacks a3′ hydroxyl group; a nucleotide that has been modified to contain ablocking group at the 3′ position; and/or a nucleotide that has beenmodified with a 3′-O-azido group, a 3′-O-azidomethyl group, a 3T-O-alkylhydroxylamino group, a 3′-phosphorothioate group, a 3′-O-malonyl group,or a 3′-O-benzyl group.

In some instances, the polymer core may comprise a linear or branchedpolymer, e.g., linear or branched polyethylene glycol (PEG),polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolicacid, poly-glycine, polyvinyl acetate, a dextran, a protein, or othersuch polymers, or copolymers incorporating any two or more of theforegoing, or incorporating other polymers as are known in the art. Insome instances, the polymer is a PEG. In some instances, the polymer isa branched PEG. In some instances, a branched polymer may comprise 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more branches or arms,or 2, 4, 8, 16, 32, 64, or more, branches or arms. In some instances,the branches or arms may radiate from a central moiety.

In some instances, the polymer-nucleotide conjugate may further compriseone or more detectable labels, e.g., one, two, three, four, five, six,seven, eight, nine, ten, fifteen, twenty, or more than twenty detectablelabels. In some instances, the one or more detectable labels maycomprise one or more fluorophores (e.g., cyanine dye 3 (Cy3), cyaninedye 5 (Cy5), etc.), one or more quantum dots, a fluorescence resonanceenergy transfer (FRET) donor, and/or a FRET acceptor.

In some instances, the polymer-nucleotide conjugate may further comprisea binding moiety attached to each branch of the polymer core or to asubset of branches. Examples of suitable binding moieties include, butare not limited to, biotin, avidin, streptavidin, or the like,polyhistidine domains, complementary paired nucleic acid domains,G-quartet forming nucleic acid domains, calmodulin, maltose-bindingprotein, cellulase, maltose, sucrose, glutathione-S-transferase,glutathione, 0-6-methylguanine-DNA methyltransferase, benzylguanine andderivatives thereof, benzylcysteine and derivatives thereof, anantibody, an epitope, a protein A, or a protein G. The binding moietymay be any interactive molecule or fragment thereof known in the art tobind to or facilitate interactions between proteins, between proteinsand ligands, between proteins and nucleic acids, between nucleic acids,or between small molecule interaction domains or moieties.

As noted above, in the sequencing-by-avidity approach a multivalentbinding complex is formed between, e.g., a fluorescently-labeledpolymer-nucleotide conjugate, a polymerase, and a plurality of primedtarget nucleic acid molecules tethered to a sample support structure(e.g., a flow cell surface) when the nucleotide moieties of thepolymer-nucleotide conjugate are complementary to a nucleotide residueof the target sequence. The stability of the multivalent binding complexthus formed allows the detection/base-calling step in a sequencingreaction cycle to be separated from the nucleotide incorporation step.

The stability of the multivalent binding complex - a ternary complexformed between two or more nucleotide moieties of the polymer-nucleotideconjugate, two or more polymerase molecules, and two or more primedtarget nucleic acid sequences—is evidenced by the prolonged persistencetimes of the complex. For example, in some instances, said multivalentbinding complexes (ternary complexes) may have a persistence time ofless than 0.5 seconds, less than 1 second, greater than 1 second,greater than 2 seconds, greater than 3 seconds, greater than 4 seconds,greater than 5 seconds, greater than 10 seconds, greater than 15seconds, greater than 20 seconds, greater than 30 seconds, greater than60 seconds, greater than 120 seconds, greater than 360 seconds, greaterthan 720 seconds, greater than 1,440 seconds, greater than 3,600seconds, or more, or for a time within a range defined by any two ormore of these values.

The use of polymer-nucleotide conjugates to form a multivalent bindingcomplex with the polymerase and primed target nucleic acid results in aneffective local concentration of the nucleotide that is increased manyfold over the average nucleotide concentration that would be achievedusing single unconjugated or untethered nucleotides, which in turn bothenhances the stability of the complex and increases signal intensityfollowing wash steps. The high signal intensity persists throughout thebinding, washing, and imaging steps, and contributes to shorter imageacquisition times. Following the imaging step, the multivalent bindingcomplex can be destabilized, e.g., by changing the ionic composition,ionic strength, and/or the pH of the buffer, and washed away. A primerextension reaction may then be performed to extend the complementarystrand by one base.

Nucleic acid sequencing system performance: In some instances, thedisclosed nucleic acid sequencing systems, comprising one or more of thedisclosed flow cell devices used in combination with one or more of thedisclosed optical imaging systems, and optionally utilizing one of theemerging sequencing biochemistries such as the “sequencing-by-trapping”(or “sequencing-by-avidity”) approach described above, may provideimproved nucleic acid sequencing performance in terms of, e.g., reducedsample input requirements, reduced image acquisition cycle time, reducedsequencing reaction cycle time, reduced sequencing run time, improvedbase-calling accuracy, reduced reagent consumption and cost, highersequencing throughput, and reduced sequencing cost.

Nucleic acid sample input (pM): In some instances, the sample inputrequirements for the disclosed system may be significantly reduced dueto the improved hybridization and amplification efficiencies that may beattained, and the high CNR images that may be acquired for base-calling,using the disclosed hydrophilic, polymer coated flow cell devices andimaging systems. In some instances, the nucleic acid sample inputrequirement for the disclosed systems may range from about 1 pM to about10,000 pM. In some instances, the nucleic acid sample input requirementmay be at least 1 pM, at least 2 pM, at least 5 pM, at least 10 pM, atleast 20 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least500 pM, at least 1,000 pM, at least 2,000 pM, at least 5,000 pM, atleast 10,000 pM. In some instances, the nucleic acid sample inputrequirement for the disclosed systems may be at most 10,000 pM, at most5,000 pM, at most 2,000 pM, at most 1,000 pM, at most 500 pM, at most200 pM, at most 100 pM, at most 50 pM, at most 20 pM, at most 10 pM, atmost 5 pM, at most 2 pM, or at most 1 pM. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe nucleic acid sample input requirement for the disclosed systems mayrange from about 5 pM to about 500 pM. Those of skill in the art willrecognize that the nucleic acid sample input requirement may have anyvalue within this range, e.g., about 132 pM. In one exemplary instance,a nucleic acid sample input of about 100 pM is sufficient to generatesignals for reliable base-calling.

Nucleic acid sample input (nanograms): In some instances, the nucleicacid sample input requirement for the disclosed systems may range fromabout 0.05 nanograms to about 1,000 nanograms. In some instances, thenucleic acid sample input requirement may be at least 0.05 nanograms, atleast 0.1 nanograms, at least 0.2 nanograms, at least 0.4 nanograms, atleast 0.6 nanograms, at least 0.8 nanograms, at least 1.0 nanograms, atleast 2 nanograms, at least 4 nanograms, at least 6 nanograms, at least8 nanograms, at least 10 nanograms, at least 20 nanograms, at least 40nanograms, at least 60 nanograms, at least 80 nanograms, at least 100nanograms, at least 200 nanograms, at least 400 nanograms, at least 600nanograms, at least 800 nanograms, or at least 1,000 nanograms. In someinstances, the nucleic acid sample input requirement may be at most1,000 nanograms, at most 800 nanograms, at most 600 nanograms, at most400 nanograms, at most 200 nanograms, at most 100 nanograms, at most 80nanograms, at most 60 nanograms, at most 40 nanograms, at most 20nanograms, at most 10 nanograms, at most 8 nanograms, at most 6nanograms, at most 4 nanograms, at most 2 nanograms, at most 1nanograms, at most 0.8 nanograms, at most 0.6 nanograms, at most 0.4nanograms, at most 0.2 nanograms, at most 0.1 nanograms, or at most 0.05nanograms. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the nucleic acid sample input requirementfor the disclosed systems may range from about 0.6 nanograms to about400 nanograms. Those of skill in the art will recognize that the nucleicacid sample input requirement may have any value within this range,e.g., about 2.65 nanograms.

# FOV images required to tile flow cell: In some instances, thefield-of-view (FOV) of the disclosed optical imaging module issufficiently large that a multi-channel (or multi-lane) flow cell (i.e.,the fluid channel portions thereof) of the present disclosure may beimaged by tiling from about 10 FOV images (or “frames”) to about 1,000FOV images (or “frames”). In some instances, an image of the entiremulti-channel flow cell may require tiling at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 300, at least 350, at least 400, at least 450, at least 500, atleast 550, at least 600, at least 650, at least 700, at least 750, atleast 800, at least 850, at least 900, at least 950, or at least 1,000FOV images (or “frames”). In some instances, an image of the entiremulti-channel flow cell may require tiling at most 1,000, at most 950,at most 900, at most 850, at most 800, at most 750, at most 700, at most650, at most 600, at most 550, at most 500, at most 450, at most 400, atmost 350, at most 300, at most 250, at most 200, at most 150, at most100, at most 90, at most 80, at most 80, at most 70, at most 60, at most50, at most 40, at most 30, at most 20, or at most 10 FOV images (or“frames”). Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances an image of the entire multi-channel flowcell may require tiling from about 30 to about 100 FOV images. Those ofskill in the art will recognize that in some instances the number ofrequired FOV images may have any value within this range, e.g., about 54FOV images.

Imaging cycle time: In some instances, the combination of large FOV,image sensor response sensitivity, and/or fast FOV translation timesenables shortened imaging cycle times (i.e., the time required toacquire a sufficient number of FOV images to tile the entiremultichannel flow cell (or the fluid channel portions thereof). In someinstances, the imaging cycle time may range from about 10 seconds toabout 10 minutes. In some instances, the imaging cycle time may be atleast 10 seconds at least 20 seconds, at least 30 seconds, at least 40seconds, at least 50 seconds, at least 1 minute, at least 2 minutes, atleast 3 minutes, at least 4 minutes, at least 5 minutes, at least 6minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, orat least 10 minutes. In some instances, the imaging cycle time may be atmost 10 minutes, at most 9 minutes, at most 8 minutes, at most 7minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, atmost 3 minutes, at most 2 minutes, at most 1 minute, at most 50 second,at most 40 second, at most 30 seconds, at most 20 seconds, or at most 10seconds. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the imaging cycle time may range fromabout 20 seconds to about 1 minute. Those of skill in the art willrecognize that in some instances the imaging cycle time may have anyvalue within this range, e.g., about 57 seconds.

Sequencing cycle time: In some instances, shortened sequencing reactionsteps, e.g., due to reduced wash time requirements for the disclosedhydrophilic, polymer-coated flow cells, may result in shortened overallsequencing cycle times. In some instances, the sequencing cycle timesfor the disclosed systems may range from about 1 minute to about 60minutes. In some instances, the sequencing cycle time may be at least 1minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, atleast 5 minutes, at least 6 minutes, at least 7 minutes, at least 8minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes,at least 20 minutes, at least 25 minutes, at least 30 minutes, at least35 minutes, at least 40 minutes, at least 45 minutes, at least 50minutes, at least 55 minutes, or at least 60 minutes. In some instances,the sequencing reaction cycle time may be at most 60 minutes, at most 55minutes, at most 50 minutes, at most 45 minutes, at most 40 minutes, atmost 35 minutes, at most 30 minutes, at most 25 minutes, at most 20minutes, at most 15 minutes, at most 10 minutes, at most 9 minutes, atmost 8 minutes, at most 7 minutes, at most 6 minutes, at most 5 minutes,at most 4 minutes, at most 3 minutes, at most 2 minutes, or at most 1minutes. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, in some instances the sequencing cycle time may range fromabout 2 minutes to about 15 minutes. Those of skill in the art willrecognize that in some instances the sequencing cycle time may have anyvalue within this range, e.g., about 1 minute, 12 seconds.

Sequencing read length: In some instances, the enhanced CNR images thatmay be achieved using the disclosed hydrophilic, polymer-coated flowcell devices in combination with the disclosed imaging systems, and insome cases, the use of milder sequencing biochemistries, may enablelonger sequencing read lengths for the disclosed systems. In someinstances, the maximum (single read) read length may range from about 50bp to about 500 bp. In some instances, the maximum (single read) readlength may be at least 50 bp, at least 100 bp, at least 150 bp, at least200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400bp, at least 450 bp, or at least 500 bp. In some instances, the maximum(single read) read length is at most 500 bp, at most 450 bp, at most 400bp, at most 350 bp, at most 300 bp, at most 250 bp, at most 200 bp, atmost 150 bp, at most 100 bp, or at most 50 bp. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe maximum (single read) read length may range from about 100 bp toabout 450 bp. Those of skill in the art will recognize that in someinstances the maximum (single read) read length may have any valuewithin this range, e.g., about 380 bp.

Sequencing run time: In some instances, the sequencing run time for thedisclosed nucleic acid sequencing systems may range from about 8 hoursto about 20 hours. In some instances, the sequencing run time is atleast 8 hours, at least 9 hours, at least 10 hours, at least 12 hours,at least 14 hours, at least 16 hours, at least 18 hours, or at least 20hours. In some instances, the sequencing run time is at most 20 hours,at most 18 hours, at most 16 hours, at most 14 hours, at most 12 hours,at most 10 hours, at most 9 hours, or at most 8 hours. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the sequencing run time may range from about 10 hours to about16 hours. Those of skill in the art will recognize that in someinstances the sequencing run time may have any value within this range,e.g., about 7 hours, 35 minutes.

Average base-calling accuracy: In some instances, the disclosed nucleicacid sequencing systems may provide an average base-calling accuracy ofat least 80%, at least 85%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9% correct over the course of a sequencing run. In someinstances, the disclosed nucleic acid sequencing systems may provide anaverage base-calling accuracy of at least 80%, at least 85%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99%, at least 99.5%, at least 99.8%, or at least 99.9% correct per every1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or100,000 bases called.

Average Q-score: In some instances, the quality or accuracy of asequencing run may be assessed by calculating a Phred quality score(also referred to as a quality score or “Q-score”), which indicates theprobability that a given base is called incorrectly by the sequencingsystem. For example, in some instances base calling accuracy for aspecific sequencing chemistry and/or sequencing system may be assessedfor a large empirical data set derived from performing sequencing runson a library of known nucleic acid sequences. The Q-score may then becalculated according to the equation:

Q=−10 log₁₀ P

where P is the base calling error probability. A Q-score of 30, forexample, indicates a probability of making a base calling error of 1 inevery 1000 bases called (or a base calling accuracy of 99.9%).

In some instances, the disclosed nucleic acid sequencing systems mayprovide a more accurate base readout. In some instances, for example,the disclosed nucleic acid sequencing systems may provide a Q-score forbase-calling accuracy over a sequencing run that ranges from about 20 toabout 50. In some instances, the average Q-score for the run may be atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, or at least 50. Those of skill in the art will recognize that theaverage Q-score may have any value within this range, e.g., about 32.

Q-score vs. % nucleotides identified: In some instances, the disclosednucleic acid sequencing systems may provide a Q-score of greater than 20for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some instances, thedisclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 25 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 30 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 35 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 40 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed nucleic acid sequencing systems may provide a Q-score ofgreater than 45 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed compositions and methods for nucleic acid sequencing mayprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

Reagent consumption: In some instances, the disclosed nucleic acidsequencing systems may have lower reagent consumption rates and costsdue to, e.g., the use of the disclosed flow cell devices and fluidicsystems that minimize fluid channel volumes and dead volumes. In someinstances, the disclosed nucleic acid sequencing systems may thusrequire an average of at least 5% less, at least 10% less, at least 15%less, at least 20% less, at least 25% less, at least 30% less, at least35% less, at least 40% less, at least 45% less, or at least 50% lessreagent by volume per Gbase sequenced that that consumed by an IlluminaMiSeq sequencer.

Sequencing throughput: In some instances, the disclosed nucleic acidsequencing systems may provide a sequencing throughput ranging fromabout 50 Gbase/run to about 200 Gbase/run. In some instances, thesequencing throughput may be at least 50 Gbase/run, at least 75Gbase/run, at least 100 Gbase/run, at least 125 Gbase/run, at least 150Gbase/run, at least 175 Gbase/run, or at least 200 Gbase/run. In someinstances, the sequencing throughput may be at most 200 Gbase/run, atmost 175 Gbase/run, at most 150 Gbase/run, at most 125 Gbase/run, atmost 100 Gbase/run, at most 75 Gbase/run, or at most 50 Gbase/run. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the sequencing throughput may range fromabout 75 Gbase/run to about 150 Gbase/run. Those of skill in the artwill recognize that in some instances the sequencing throughput may haveany value within this range, e.g., about 119 Gbase/run.

Sequencing cost: In some instances, the disclosed nucleic acidsequencing systems may provide nucleic acid sequencing at a cost rangingfrom about $5 per Gbase to about $30 per Gbase. In some instances, thesequencing cost may be at least $5 per Gbase, at least $10 per Gbase, atleast $15 per Gbase, at least $20 per Gbase, at least $25 per Gbase, orat least $30 per Gbase. In some instances, the sequencing cost may be atmost $30 per Gbase, at most $25 per Gbase, at most $20 per Gbase, atmost $15 per Gbase, at most $10 per Gbase, or at most $30 per Gbase. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the sequencing cost may range from about $10per Gbase to about $15 per Gbase. Those of skill in the art willrecognize that in some instances the sequencing cost may have any valuewithin this range, e.g., about $7.25 per Gbase.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Design Specifications for a Fluorescence Imaging Module ForGenomics Applications

A non-limiting example of design specifications for a fluorescenceimaging module of the present disclosure is provided in Table 1.

TABLE 1 Examples of design specifications for a fluorescence imagingmodule for genomics applications. Design Parameter SpecificationNumerical aperture ≥0.3 Image quality Diffraction limited Field-of-view(FOV) >2.0 mm² Image plane curvature Best focal plane within 100 nmfor >90% of the FOV, within 150 nm for 99% of the FOV, and within 200 nmfor the entire FOV Image distortion <0.5% across the FOV Magnification2x to 20x Camera pixel size at ≥2x optical system modulation sampleplane transfer function (MTF) limit Coverslip thickness >700 μm Numberof fluorescence ≥3 imaging channels Chromatic focal plane ≤100 nmequivalent at sample difference at camera plane between all imagingchannels Number of AF channels 1 Imaging time ≤2 seconds per FOVAutofocus Single step autofocus with error correction Autofocus accuracy<100 nm Scanning stage step and <0.4 seconds settle timeChannel-specific 1 per imaging channel optimized tube lens Illuminationoptical path Liquid light guide with underfilled entrance aperture

Example 2 Fabrication of Glass Microfluidic Flow Cell Devices

Wafer-scale fabrication of microfluidic devices for use as flow cellscan be constructed from, for example, one, two, or three layers ofglass, e.g., borosilicate glass, fused-silica glass, or quartz, usingone of the processed illustrated in FIGS. 36A-36C and a processingtechnique such as focused femtosecond laser photoablation and/or laserglass bonding.

In FIG. 36A, a first wafer is processed with a laser (e.g., thatproduces femtosecond laser radiation) to ablate the wafer material andprovide a patterned surface. The patterned wafer surface may comprise aplurality of microfluidic devices (e.g., 12 devices per 210 mm diameterwafer), each of which may comprise a plurality of fluid channels. Theprocessed wafer may then be diced to create individual microfluidicchips comprising open fluid channels that may optionally be subsequentlysealed, e.g., by sealing with a film or by clamping the device toanother support surface.

In FIG. 36B, a first wafer is processed to create a patterned surfacewhich may then be placed in contact with and bonded to a second wafer toseal the fluid channels. Depending on the materials used, e.g., glasswafers, silicon wafers, etc., the bonding may be performed using, e.g.,a thermal bonding process, an anodic bonding process, a laser glassbonding process, etc. The second wafer covers and/or seals the grooves,indentations, and/or apertures on the wafer having the patterned surfaceto form fluid channels and/or fluid chambers (e.g., the interiorportion) of the device at the interface of the two wafer components. Thebonded structure may then be diced into individual microfluidic chips,e.g., 12 microfluidic chips per 210 mm diameter wafer.

In FIG. 36C, the first wafer is processed to create a pattern of fluidchannels that are cut or etched through the full thickness of the wafer(i.e., open on either surface of the wafer). The first wafer is thensandwiched between and bonded to a second wafer on one side and a thirdwafer on the other side. Depending on the materials used, e.g., glasswafers, silicon wafers, etc., the bonding may be performed using, e.g.,a thermal bonding process, an anodic bonding process, a laser glassbonding process, etc. The second and third wafers cover and/or seal thegrooves, indentations, and/or apertures in the first wafer to form fluidchannels and/or fluid chambers (e.g., the interior portions) of thedevice. The bonded structure may then be diced into individualmicrofluidic chips, e.g., 12 microfluidic chips per 210 mm diameterwafer.

Example 3 Coating Flow Cell Surfaces with a Hydrophilic Polymer Coating

Glass flow cell devices were coated by washing prepared glass channelswith KOH, followed by rinsing with ethanol and then silanization for 30minutes at 65° C. Fluid channel surfaces were activated with EDC-NHS for30 min., followed by grafting of oligonucleotide primers by incubationof the activated surface with 5 μm primer for 20 min., and thenpassivation with 30 μm of an amino-terminated polyethylene glycol(PEG-NH₂).

Multilayer surfaces are made following the approach described above,where following the PEG-NH₂ passivation step, a multi-armed PEG-NHS isflowed through the fluid channels, followed by another addition of thePEG-NH₂, optionally followed by another incubation with PEG-NHS, andoptionally followed by another incubation with multi-armed PEG-NH₂. Forthese surfaces, the primer may be grafted at any step, and especiallyfollowing the last addition of multi-armed PEG-NH₂.

Example 4 Flow Cell Devices for Nucleic Acid Sequencing

FIG. 37A illustrates a non-limiting example of a one-piece glassmicrofluidic chip/flow cell design. In this design, fluid channels andinlet/outlet holes may be fabricated using, e.g., focused femtosecondlaser radiation. There are two fluid channels (“lanes”) in the flow celldevice, and each fluid channel comprises, e.g., 2 rows of 26 frames each(i.e., where a “frame” is the image area equivalent to the field-of-viewfor a corresponding imaging module) each, such that tiling 2×26=52images suffices to image an entire fluid channel. The fluid channel canhave, e.g., a depth of about 100 μm. Fluid channel 1 has an inlet holeAl and an outlet hole A2, and fluid channel 2 has an inlet hole B1 andan outlet hole B2. The flow cell device may also comprise a 1D linear,human-readable and/or machine-readable barcode, and optionally a 2Dmatrix barcode.

FIG. 37B illustrates a non-limiting example of a two-piece glassmicrofluidic chip/flow cell design. In this design, fluid channels andinlet/outlet holes may be fabricated using, e.g., focused femtosecondlaser photoablation or photolithography and chemical etching processes.The 2 pieces can be bonded together using any of a variety of techniquesas described above. The inlet and outlet holes may be positioned on thetop layer of the structure and oriented in such a way that they are influid communication with at least one of the fluid channels and/or fluidchambers formed in the interior portion of the device. There are twofluid channels in the flow cell device, and as with the deviceillustrated in FIG. 37A, each fluid channel comprises, e.g., 2 rows with26 frames in each row. The fluid channels can have, e.g., a depth ofabout 100 μm. Fluid channel 1 has an inlet hole A1 and an outlet holeA2, and fluid channel 2 has an inlet hole B1 and an outlet hole B2. Theflow cell device may also comprise a 1D linear, human-readable and/ormachine-readable barcode, and optionally a 2D matrix barcode.

FIG. 37C illustrates a non-limiting example of a three-piece glassmicrofluidic chip/flow cell design. In this design, fluid channels andinlet/outlet holes may be fabricated using, e.g., focused femtosecondlaser photoablation or photolithography and chemical etching processes.The 3 pieces can be bonded together using any of a variety of techniquesas described above. The first wafer (comprising a through-pattern offluid channels or fluid chambers) can be sandwiched between and bondedto a second wafer on one side and a third wafer on the other side. Theinlet and outlet holes may be positioned on the top layer of thestructure and oriented in a way such that they are in fluidcommunication with at least one of the fluid channels and/or fluidchambers formed in the interior portion of the device. There are twofluid channels in the flow cell device, and as with the devicesillustrated in FIGS. 37A and 37B, each fluid channel has 2 rows with 26frames in each row. The fluid channel can have a depth of, e.g., about100 μm. Fluid channel 1 has an inlet hole Al and an outlet hole A2, andfluid channel 2 has an inlet hole B1 and an outlet hole B2. The flowcell device may also comprise a 1D linear, human-readable and/ormachine-readable barcode, and optionally a 2D matrix barcode.

Example 5 Imaging of Nucleic Acid Clusters in a Capillary Flow Cell

Nucleic acid clusters were established within a capillary and subjectedto fluorescence imaging. A flow device having a capillary tube was usedfor the test. An example of the resulting cluster images is presented inFIG. 38. The figure demonstrated that nucleic acid clusters formed byamplification within the lumen of a capillary flow cell device asdisclosed herein can be reliably formed and visualized.

Example 6 Plastic Sample Support Structures

In some instances, the disclosed samples support structures may befabricated from a polymer. Examples of materials from which the samplesupport structure, e.g., a capillary flow cell device, may be fabricatedinclude, but are not limited to, polystyrene (PS), macroporouspolystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC),polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),cyclic olefin polymers (COP), cyclic olefin copolymers (COC),polyethylene terephthalate (PET), or any combination thereof. Variouscompositions comprising both glass and plastic substrates are alsocontemplated.

Modification of a polymer surface for the surface coating purposesdisclosed herein involves making surfaces reactive with other chemicalgroups (-R), including amines. When prepared on an appropriatesubstrate, these reactive surfaces can be stored long term at roomtemperature, for example, for at least 3 months or more in someinstances. Such surfaces can be further grafted with R-PEG and R-primeroligomer for on-surface amplification of nucleic acids, as describedelsewhere herein. Plastic surfaces, such as cyclic olefin polymer (COP),may be modified using any of a variety of methods known in the art. Forexample, they can be treated with Ti:Sapphire laser ablation,UV-mediated ethylene glycol methacrylate photografting, plasmatreatment, or mechanical agitation (e.g., sand blasting, or polishing,etc.) to create hydrophilic surfaces that can remain reactive for monthstowards a variety of chemical groups, such as amines. These groups maythen allow conjugation of passivation polymers such as PEG, orbiomolecules such as DNA or proteins, without loss of biochemicalactivity. For example, attachment of DNA primer oligomers allows DNAamplification on a passivated plastic surface while reducing orminimizing the non-specific adsorption of proteins, fluorophoremolecules, or other hydrophobic molecules.

Additionally, in some instances, surface modification can be combinedwith, e.g., laser printing or UV masking, to create patterned surfaces.This allows patterned attachment of DNA oligomers, proteins, or othermoieties, providing for surface-based enzymatic activity, binding,detection, or processing. For example, DNA oligomers may be used toamplify DNA only within patterned features, or to capture amplified longDNA concatemers in a patterned fashion. In some embodiments, enzymeislands may be generated in the patterned areas that are capable ofreacting with solution-based substrates. Because plastic surfaces areespecially amenable to these processing modes, in some embodiments ascontemplated herein, plastic sample support surfaces or flow celldevices may be recognized as being particularly advantageous.

Furthermore, plastic can be injection molded, embossed, stamped, or 3Dprinted to form any shape, including microfluidic devices, much moreeasily than glass substrates, and thus can be used to create surfacesfor the binding and analysis of biological samples in multipleconfigurations, e.g., sample-to-result microfluidic chips for biomarkerdetection or DNA sequencing.

Specific and localized DNA amplification on modified plastic surfacescan be performed to produce nucleic acid spots with an ultra-highcontrast to noise ratio and very low background when probed withfluorescent labels.

Hydrophilized and amine-reactive cyclic olefin polymer surface withamine-primer and amine-PEG can be prepared and has been demonstrated tosupport rolling circle amplification. When probed with fluorophorelabeled primers, or when labeled dNTPs were added to the hybridizedprimers by a polymerase, bright spots of DNA amplicons were observedthat exhibited signal to noise ratios greater than 100 with backgroundsthat are extremely low, indicating highly specific amplification, andultra-low levels of nonspecific protein and hydrophobic fluorophorebinding, which are hallmarks of the high accuracy detection required forsystems such as fluorescence-based DNA sequencers.

Example 7 Prophetic Example of the Use of a Structured IlluminationImaging System for Sequencing

A structured illumination imaging system 4100 such as the non-limitingexample illustrated in FIG. 41 may be used in combination with a flowcell 4187 comprising a low non-specific binding surface to performnucleic acid sequencing. Target nucleic acid sequences are hybridized toadapter/primer sequences attached to the low non-specific bindingsurface 4188 on the interior of the flow cell 4187 at high surfacedensity and clonally amplified using hybridization and amplificationbuffers that are specially formulated for said surface to enhancespecific hybridization and amplification rates.

The flow cell 4187 is mounted in the structured illumination imagingsystem 4100, and a sequencing reaction cycle comprising the use of,e.g., the polymer-nucleotide conjugate chemistry described above and theworkflow illustrated in FIG. 40 is initiated. The fluorescently labeledpolymer nucleotide conjugate is introduced into the flow cell 4187 andcontacted with the surface 4188 to form multivalent binding complexes ifthe nucleotide moiety of the polymer-nucleotide conjugate iscomplementary to a nucleotide of the target sequence. Excess, unboundpolymer-nucleotide conjugate is then rinsed away.

For each detection step, a series of images of surface 4188 are capturedusing different orientations of a diffraction grating, e.g., 4130A, inat least one branch of an illumination optical path and at severaldifferent positions of an optical phase modulator, e.g., 4140A, toproject illumination light fringe patterns onto the surface 4188.Following image acquisition, the series of images are processed using animage reconstruction algorithm to generate a higher resolution imagethan that achievable using diffraction-limited optics alone. The processmay be repeated for several positions on surface 4188 to create a tiledimage of the interior flow cell surface. Optionally, the focal plane maybe adjusted, and the process may be repeated to generate higherresolution images of a second interior flow cell surface 4189.

The combination of high contrast-to-noise ratio images (achieved usingthe disclosed low-binding surfaces with multiply-labeledpolymer-nucleotide conjugate sequencing chemistry) and efficientprocessing of a relatively small number of images acquired using astructured illumination imaging system to image flow cell surfaces atsuper-resolution (thus enabling the use of higher surface densities oftarget sequence clusters) may contribute to higher overall sequencingthroughput.

Example 8 Prophetic Example of Using a Multiplexed Read-Head for DualSurface Imaging

A multiplexed read-head such as that illustrated schematically in FIGS.44A and 44B is designed to perform dual surface imaging. The read-headcomprises a plurality of microfluorometers which are assembled so thatthey are held in a fixed positions relative to one another and may bescanned in a direction horizontal to a pair of opposed interior flowcell surfaces to acquire images of a swath of each surface. Asillustrated in FIG. 44A, a first subset of the plurality ofmicrofluorometers is configured to acquire images of a first interiorflow cell surface, and a second subset of the plurality ofmicrofluorometers is configured to acquire images of a second interiorflow cell surface that faces the first interior surface and is separatedfrom it by the thickness of an intervening fluid channel.

A flow cell comprising a low non-specific binding surface coating isused to perform nucleic acid sequencing. Target nucleic acid sequencesare hybridized to adapter/primer sequences attached to the lownon-specific binding surfaces on the interior of the flow cell andclonally amplified using hybridization and amplification buffers thatare specially formulated for said surfaces to enhance specifichybridization and amplification rates.

The flow cell is mounted in an imaging system comprising the multiplexedread-head, and a sequencing reaction cycle comprising the use of, e.g.,the polymer-nucleotide conjugate chemistry described above and theworkflow illustrated in FIG. 40 is initiated. The fluorescently labeledpolymer nucleotide conjugate is introduced into the flow cell andcontacted with the interior surfaces to form multivalent bindingcomplexes if the nucleotide moiety of the polymer-nucleotide conjugateis complementary to a nucleotide of the target sequence. Excess, unboundpolymer-nucleotide conjugate is then rinsed away.

For each detection step, the multiplexed read-head is scanned in atleast one direction parallel to the interior surfaces of the flow cell(or the flow cell may be scanned relative to the multiplexed read-head)and images of both the first and second interior flow cell surfaces areacquired simultaneously, as illustrated in FIG. 44B, while an autofocusmechanism maintains the proper working distance between the objectivesof the multiplexed read-head and at least one of the interior flow cellsurfaces.

The ability to image both flow cell surfaces simultaneously using asingle-pass scan of the flow cell (depending on the design of theread-head) may provide significant improvements in sequencingthroughput.

Additional Numbered Embodiments

-   1. A system for sequencing nucleic acid molecules comprising:    -   a) a flow cell with an interior surface comprising a plurality        of primed target nucleic acid sequences coupled thereto, wherein        a primed target nucleic acid sequence of the plurality of primed        target nucleic acid sequences has a polymerase bound thereto;    -   b) a fluid flow controller configured to control sequential and        iterative delivery of a reagent to the interior surface of the        flow cell;    -   c) an imaging module comprising:        -   i) a structured illumination system; and        -   ii) an image acquisition system configured to acquire images            of the interior surface of the flow cell; and    -   d) a processor, wherein the processor is programed to instruct        the system to perform an iterative method comprising:        -   i) contacting the plurality of primed target nucleic acid            sequences coupled to the interior surface of the flow cell            with a nucleotide composition to form a transient binding            complex between the plurality of primed target nucleic acid            sequences and a plurality of nucleotide moieties when a            nucleotide moiety of the nucleotide composition is            complementary to a nucleotide of the primed target nucleic            acid sequence; and        -   ii) imaging the interior surface of the flow cell to detect            the transient binding complex and thereby determine an            identity of the nucleotide of the primed target nucleic acid            sequence.-   2. The system of claim 1, wherein the structured illumination system    comprises an optical system designed to project periodic patterns of    light on the interior surface of the flow cell, and wherein a    relative orientation or phase shift of a plurality of the periodic    patterns of light may be changed in a known manner.-   3. The system of claim 1, wherein the structured illumination system    comprises a first optical arm comprising a first light emitter to    emit light and a first beam splitter to split light emitted by the    first light emitter to project a first plurality of fringes on the    interior surface of the flow cell.-   4. The system of claim 3, wherein the structured illumination system    further comprises a second optical arm comprising a second light    emitter to emit light and a second beam splitter to split light    emitted by the second light emitter to project a second plurality of    fringes on the interior surface of the flow cell.-   5. The system of claim 4, wherein the structured illumination system    further comprises an optical element to combine an optical path of    the first arm and the second arm.-   6. The system of claim 4 or claim 5, wherein the first beam splitter    comprises a first transmissive diffraction grating and the second    beam splitter comprises a second transmissive diffraction grating.-   7. The system of claim 4 or claim 5, wherein the first and second    light emitters emit unpolarized light, and wherein the first and    second transmissive diffraction gratings are to diffract unpolarized    light emitted by a respective one of the first and second light    emitters.-   8. The system of claim 6 or claim 7, wherein the optical element to    combine an optical path of the first plurality of fringes and the    second plurality of fringes comprises a mirror with holes, with the    mirror arranged to reflect light diffracted by the first    transmissive diffraction grating and with the holes arranged to pass    through at least first orders of light diffracted by the second    transmissive diffraction grating.-   9. The system of claim 8, further comprising: one or more optical    elements to phase shift the first plurality of fringes and the    second plurality of fringes.-   10. The system of claim 9, wherein the one or more optical elements    to phase shift the first plurality of fringes and the second    plurality of fringes comprise a first rotating optical window to    phase shift the first plurality of fringes and a second rotating    optical window to phase shift the second plurality of optical    fringes.-   11. The system of claim 9 or claim 10, wherein the one or more    optical elements to phase shift the first plurality of fringes and    the second plurality of fringes comprise a first linear motion stage    to translate the first diffraction grating and a second linear    motion stage to translate the second diffraction grating.-   12. The system of any one of claims 9 to 11, wherein the one or more    optical elements to phase shift the first plurality of fringes and    the second plurality of fringes comprise a single rotating optical    window, wherein the single rotating optical window is positioned    after the mirror with holes in an optical path to the sample.

13. The system of claim 12, wherein an axis of rotation of the singlerotating optical window is offset by about 45 degrees from an opticalaxis of each of the gratings.

14. The system of any one of claims 9 to 13, wherein the first pluralityof fringes are angularly offset from the second plurality of fringes onthe sample plane by about 90 degrees.

-   15. The system of claim 14, wherein the sample comprises a plurality    of features regularly patterned in a rectangular array or hexagonal    array.-   16. The system of any one of claims 9 to 15, further comprising: an    objective lens to project each of the first plurality of fringes and    the second plurality of fringes on the sample.-   17. The system of any one of claims 9 to 16, further comprising: one    or more optical beam blockers for blocking zero orders of light    emitted by each of the first and second diffraction gratings.-   18. The system of claim 17, wherein the one or more optical beam    blockers comprise a Bragg grating.-   19. The system of any one of claims 6 to 18, wherein the optical    element to combine an optical path of the first arm and the second    arm comprises a polarizing beam splitter, wherein the first    diffraction grating diffracts vertically polarized light and wherein    the second diffraction grating diffracts horizontally polarized    light.-   20. The system of any one of claims 4 to 19, wherein the first and    second beam splitters each comprise a beam splitter cube or plate.-   21. The system of any one of claims 3 to 20, wherein the first beam    splitter comprises a first reflective diffraction grating and the    second beam splitter comprises a second reflective diffraction    grating.-   22. The system of any one of claims 1 to 21, wherein the structured    illumination system comprises a multiple beam splitter slide    comprising a plurality of beam splitters mounted on a linear    translation stage such that the plurality of beam splitters have    fixed orientations with respect to the system's optical axis.-   23. The system of claim 22, wherein the plurality of beam splitters    comprises a plurality of diffraction gratings.-   24. The system of claim 23, wherein the plurality of diffraction    gratings comprises two diffraction gratings.-   25. The system of any one of claims 1 to 24, wherein the structured    illumination system comprises a fixed two-dimensional diffraction    grating used in combination with a spatial filter wheel to project    one-dimensional diffraction patterns on the interior surface of the    flow cell.-   26. The system of any one of claims 1 to 25, wherein the image    acquisition system comprises a custom tube lens which, in    combination with an objective, enables imaging of a first interior    flow cell surface and a second interior flow cell surface with    substantially the same image resolution.-   27. The system of any one of claims 1 to 26, wherein the nucleotide    composition comprises a conjugated polymer-nucleotide composition.-   28. The system of claim 27, wherein the conjugated    polymer-nucleotide composition comprises a plurality of nucleotide    moieties conjugated to a polymer core.-   29. The system of claim 28, wherein the plurality of nucleotide    moieties comprises nucleotides, nucleotide analogs, or any    combination thereof-   30. The system of claim 28 or claim 29, wherein the plurality of    nucleotide moieties comprises a plurality of identical nucleotide    moieties.-   31. The system of any one of claims 1 to 30, wherein prior to    forming the transient binding complex the nucleotide composition    lacks a polymerase.-   32. A method for sequencing nucleic acid molecules comprising:    -   a) providing a plurality of primed target nucleic acid sequences        tethered to a surface, wherein a primed target nucleic acid        sequence of the plurality of primed target nucleic acid        sequences has a polymerase bound thereto;    -   b) contacting the plurality of primed target nucleic acid        sequences with a nucleotide composition to form a transient        binding complex between the plurality of primed target nucleic        acid sequences and a plurality of nucleotide moieties when a        nucleotide moiety of the nucleotide composition is complementary        to a nucleotide of the primed target nucleic acid sequence; and    -   c) detecting the transient binding complex to determine the        identity of the nucleotide of the primed target nucleic acid        sequence, wherein the detecting comprises:        -   i) illuminating the surface with light provided by a            structured illumination system under a first set of            illumination conditions to project a first plurality of            fringes oriented in a specific direction on the surface;        -   ii) capturing a first plurality of phase images of the            surface, wherein during capture of the first plurality of            images, the positions of the first plurality of fringes are            shifted on the surface;        -   iii) illuminating the surface with light provided by the            structured illumination system under a second set of            illumination conditions to project a second plurality of            fringes on the surface, wherein the second plurality of            fringes are angularly offset from the first plurality of            fringes on the surface; and        -   iv) capturing a second plurality of phase images of the            surface illuminated with the second plurality of fringes,            wherein during capture of the second plurality of fringes,            the positions of the second plurality of fringes are shifted            on the surface.-   33. The method of claim 32, wherein the structured illumination    system comprises a first optical arm comprising a first light    emitter to emit light and a first diffraction grating to diffract    light emitted by the first light emitter to project the first    plurality of fringes oriented in a specific direction on the    surface.-   34. The method of claim 33, wherein the structured illumination    system comprises a second optical arm comprising a second light    emitter to emit light and a second diffraction grating to diffract    light emitted by the second light emitter to project the second    plurality of fringes that are angularly offset from the first    plurality of fringes on the surface.-   35. The method of any one of claims 32 to 34, wherein the structured    illumination system comprises a multiple beam splitter slide    comprising a plurality of beam splitters mounted on a linear    translation stage such that the plurality of beam splitters have    fixed orientations with respect to the system's optical axis, and    wherein the first set of illumination conditions corresponds to a    first position of the linear translation stage and the second set of    illumination conditions corresponds to a second position of the    linear translation stage.-   36. The method of claim 35, wherein the plurality of beam splitters    comprises a plurality of diffraction gratings.-   37. The method of claim 36, wherein the plurality of diffraction    gratings comprises two diffraction gratings.-   38. The method of any one of claims 32 to 37, wherein the structured    illumination system comprises a fixed two-dimensional diffraction    grating used in combination with a spatial filter wheel to project    one-dimensional diffraction patterns on the surface, and wherein the    first set of illumination conditions corresponds to a first position    of the spatial filter wheel and the second set of illumination    conditions corresponds to a second position of the spatial filter    wheel.-   39. The method of any one of claims 34 to 38, wherein the first    diffraction grating and the second diffraction grating are    transmissive diffraction gratings, wherein the structured    illumination system comprises a mirror with holes to reflect light    diffracted by the first diffraction grating and to pass through at    least first orders of light diffracted by the second diffraction    grating.-   40. The method of any one of claims 32 to 39, further comprising:    using at least the first plurality of captured phase images and the    second plurality of captured phased images to computationally    reconstruct one or more images having higher resolution than each of    the first and second pluralities of captured phased images.-   41. The method of claim 40, wherein the first plurality of fringes    is angularly offset from the second plurality of fringes on the    surface by about 90 degrees.-   42. The method of any one of claims 32 to 41, wherein the surface    comprises a plurality of features regularly patterned in a    rectangular array or hexagonal array.-   43. The method of any one of claims 32 to 42, wherein the first    plurality of fringes and the second plurality of fringes are phase    shifted by rotating a single optical window positioned in an optical    path between the surface and each of the first and second    diffraction gratings, wherein an axis of rotation of the single    rotating optical window is offset from an optical axis of each of    the diffraction gratings.-   44. The method of any one of claims 34 to 43, wherein the first    optical arm is turned off and the second optical arm of the    structured illumination system is turned on after capturing the    first plurality of phase images.-   45. The method of any one of claims 34 to 44, wherein the first    diffraction grating and the second diffraction grating are    mechanically fixed during image capture.-   46. The method of any one of claims 32 to 45, wherein the nucleotide    composition comprises a conjugated polymer-nucleotide composition.-   47. The method of claim 46, wherein the conjugated    polymer-nucleotide composition comprises a plurality of nucleotide    moieties conjugated to a polymer core.-   48. The method of claim 47, wherein the plurality of nucleotide    moieties comprises nucleotides, nucleotide analogs, or any    combination thereof-   49. The method of claim 47 or claim 48, wherein the plurality of    nucleotide moieties comprises a plurality of identical nucleotide    moieties.-   50. The method of any one of claims 32 to 49, wherein the method is    used to determine the identity of an N+1 or terminal nucleotide of a    primer strand of the primed target nucleic acid sequence.-   51. The method of any one of claims 32 to 50, wherein prior to    forming the transient binding complex the nucleotide composition    lacks a polymerase.-   52. A detection apparatus, comprising    -   a) a read-head assembly comprising a plurality of        microfluorometers,        -   wherein the plurality of microfluorometers are held in fixed            positions relative to each other to form a multiplexed            read-head,        -   wherein at least one of a first subset of the plurality of            microfluorometers is configured to acquire a wide-field            image of a different area of a first sample plane, and        -   wherein at least one of a second subset of the plurality of            microfluorometers is configured to acquire a wide-field            images of a different area of a second sample plane.-   53. The detection apparatus of claim 52, further comprising a    translation stage configured to move the read-head assembly in at    least one direction parallel to the first and second sample planes.-   54. The detection apparatus of claim 52 or claim 53, further    comprising a sample stage configured to hold a flow cell comprising    first and second interior surfaces such that the first interior    surface is held at the first sample plane, and the second interior    surface is held at the second sample plane.-   55. The detection apparatus of any one of claims 52 to 54, wherein    at least one microfluorimeter of the plurality of microfluorimeters    is configured to acquire wide-field images having a field-of-view of    at least 1 mm.-   56. The detection apparatus of any one of claims 52 to 55, wherein    at least one microfluorimeter of the plurality of microfluorimeters    is configured to acquire wide-field images having a field-of-view of    at least 1.5 mm.-   57. The detection apparatus of any one of claims 52 to 56, wherein    at last one of the microfluorometers further comprises a dedicated    autofocus mechanism.-   58. The detection apparatus of claim 57, wherein the autofocus    mechanism for a first microfluorometer is configured to integrate    data from an autofocus mechanism for a second microfluorometer,    whereby the autofocus mechanisms for the first microfluorometer    alters a focus of the first microfluorometer based on a focus    position of the first microfluorometer and a focus position of the    second microfluorometer.-   59. The detection apparatus of any one of claims 52 to 58, wherein    an individual microfluorometer further comprises an objective, a    beam splitter and a detector, wherein the beam splitter is    positioned to direct excitation radiation from an excitation    radiation source to the objective and to direct emission radiation    from the objective to the detector.-   60. The detection apparatus of claim 59, wherein at least one    individual microfluorometer further comprises an individual    excitation radiation source.-   61. The detection apparatus of claim 59 or claim 60, wherein the    excitation radiation source directs the excitation radiation to the    objectives of two or more individual microfluorometers of the    plurality such that the two or more individual microfluorometers    share the excitation radiation source.-   62. The detection apparatus of any one of claims 59 to 61, wherein    two or more individual microfluorometers of the plurality further    comprise or share at least two excitation radiation sources.-   63. The detection apparatus of any one of claims 59 to 62, wherein    the objectives of the individual microfluorometers of the plurality    have a numerical aperture between 0.2 and 0.5.-   64. The detection apparatus of any one of claims 52 to 63, wherein    the microfluorometers of the plurality are configured to acquire    images at a resolution sufficient to distinguish features that are    less than 50 microns apart.-   65. The detection apparatus of any one of claims 52 to 64, wherein    the microfluorometers of the plurality are configured to have a    depth-of-field that is less than the separation distance between the    first and second interior surfaces of the flow cell.-   66. The detection apparatus of any one of claims 52 to 65, wherein    the first subset of the plurality of microfluorometers is configured    to acquire wide-field images at a first fluorescence emission    wavelength and the second subset of the plurality of    microfluorometers is configured to acquire wide field images at a    second fluorescence emission wavelength.-   67. A method for determining an identity of a nucleotide in a target    nucleic acid sequence comprising:    -   a) providing a plurality of primed target nucleic acid        sequences, wherein a primed target nucleic acid sequence of the        plurality of primed target nucleic acid sequences has a        polymerase bound thereto;

b) contacting the plurality of primed target nucleic acid sequences witha nucleotide composition to form a transient binding complex between theplurality of primed target nucleic acid sequences and a plurality ofnucleotide moieties when a nucleotide moiety of the nucleotidecomposition is complementary to a nucleotide of the primed targetnucleic acid sequence; and

-   -   c) detecting the transient binding complex to determine the        identity of the nucleotide of the primed target nucleic acid        sequence, wherein the detecting comprises:        -   translating a multiplexed read-head in at least one            direction parallel to a surface on which the plurality of            primed target nucleic acid sequences is tethered,        -   wherein the multiplexed read-head comprises a plurality of            microfluorometers held in fixed positions relative to each            other, and        -   wherein at least one microfluorimeter of the plurality of            microfluorometers is configured to acquire a wide-field            image of a different area of the surface than other            microfluorimeters of the plurality.

-   68. The method of claim 67, wherein the nucleotide composition    comprises a conjugated polymer-nucleotide composition.

-   69. The method of claim 68, wherein the conjugated    polymer-nucleotide composition comprises a plurality of nucleotide    moieties conjugated to a polymer core.

-   70. The method of claim 69, wherein the plurality of nucleotide    moieties comprises nucleotides, nucleotide analogs, or any    combination thereof

-   71. The method of claim 69 or claim 70, wherein the plurality of    nucleotide moieties comprises a plurality of identical nucleotide    moieties.

-   72. The method of any one of claims 67 to 71, wherein the method is    used to determine the identity of an N+1 or terminal nucleotide of a    primer strand of the primed target nucleic acid sequence.

-   73. The method of any one of claims 67 to 72, wherein prior to    forming the transient binding complex the nucleotide composition    lacks a polymerase.

-   74. The method of any one of claims 67 to 73, wherein the plurality    of primed target nucleic acid sequences is tethered to a first    interior surface and a second interior surface of a flow cell, and    wherein a first subset of the plurality of microfluorometers is    configured to acquire wide-field images of different areas of the    first interior surface of the flow cell, and a second subset of the    plurality of microfluorometers is configured to acquire wide-field    images of different areas of the second interior surface of the flow    cell.

-   75. A system for sequencing nucleic acid molecules comprising:    -   a) a flow cell having at least one interior surface comprising a        plurality of primed target nucleic acid sequences coupled        thereto, wherein a primed target nucleic acid sequence of the        plurality of primed target nucleic acid sequences has a        polymerase bound thereto;    -   b) a fluid flow controller configured to control sequential and        iterative delivery of a reagent to the at least one interior        surface of the flow cell;    -   c) an imaging module configured to image the at least one        interior surface of the flow cell, wherein the imaging module        comprises:        -   a multiplexed read-head assembly comprising a plurality of            microfluorometers held in fixed positions relative to each            other,        -   wherein at least one microfluorimeter of the plurality of            microfluorometers is configured to acquire a wide-field            image of a different area of the at least one surface than            other microfluorimeters of the plurality; and    -   d) a processor, wherein the processor is programed to instruct        the system to perform an iterative method comprising:        -   i) contacting the plurality of primed target nucleic acid            sequences coupled to the at least one interior surface of            the flow cell with a nucleotide composition to form a            transient binding complex between the plurality of primed            target nucleic acid sequences and a plurality of nucleotide            moieties when a nucleotide moiety of the nucleotide            composition is complementary to a nucleotide of the primed            target nucleic acid sequence; and        -   ii) imaging the at least one interior surface of the flow            cell using the multiplexed read-head to detect the transient            binding complex and thereby determine the identity of the            nucleotide of the primed target nucleic acid sequence.

-   76. The system of claim 75, wherein the nucleotide composition    comprises a conjugated polymer-nucleotide composition.

-   77. The system of claim 76, wherein the conjugated    polymer-nucleotide composition comprises a plurality of nucleotide    moieties conjugated to a polymer core.

-   78. The system of claim 77, wherein the plurality of nucleotide    moieties comprises nucleotides, nucleotide analogs, or any    combination thereof

-   79. The system of claim 77 or claim 78, wherein the plurality of    nucleotide moieties comprises a plurality of identical nucleotide    moieties.

-   80. The system of any one of claims 75 to 79, wherein the method is    used to determine the identity of an N+1 or terminal nucleotide of a    primer strand of the primed target nucleic acid sequence.

-   81. The system of any one of claims 75 to 80, wherein prior to    forming the transient binding complex the nucleotide composition    lacks a polymerase.

-   82. The method of any one of claims 75 to 81, wherein the plurality    of primed target nucleic acid sequences is tethered to a first    interior surface and a second interior surface of the flow cell, and    wherein a first subset of the plurality of microfluorometers is    configured to acquire wide-field images of different areas of the    first interior surface of the flow cell, and a second subset of the    plurality of microfluorometers is configured to acquire wide-field    images of different areas of the second interior surface of the flow    cell.

-   83. The system of any one of claims 75 to 82, further comprising a    translation stage configured to move the multiplexed read-head    assembly in at least one direction parallel to the first and second    sample planes.

-   84. The system of any one of claims 75 to 83, wherein at least one    microfluorimeter of the plurality of microfluorimeters is configured    to acquire wide-field images having a field-of-view of at least 1    mm.

-   85. The system of any one of claims 75 to 84, wherein at least one    microfluorimeter of the plurality of microfluorimeters is configured    to acquire wide-field images having a field-of-view of at least 1.5    mm.

-   86. The system of any one of claims 74 to 85, wherein at least one    of the microfluorometers further comprises a dedicated autofocus    mechanism.

-   87. The system of claim 86, wherein the autofocus mechanism for a    first microfluorometer is configured to integrate data from an    autofocus mechanism for a second microfluorometer, whereby the    autofocus mechanisms for the first microfluorometer alters a focus    of the first microfluorometer based on a focus position of the first    microfluorometer and a focus position of the second    microfluorometer.

-   88. The system of any one of claims 75 to 87, wherein an individual    microfluorometer of the plurality further comprises an objective, a    beam splitter and a detector, wherein the beam splitter is    positioned to direct excitation radiation from an excitation    radiation source to the objective and to direct emission radiation    from the objective to the detector.

-   89. The system of claim 88, wherein at least one individual    microfluorometer further comprises an individual excitation    radiation source.

-   90. The system of claim 89, wherein the excitation radiation source    directs the excitation radiation to the objectives of two or more    individual microfluorometers of the plurality such that the two or    more individual microfluorometers share the excitation radiation    source.

-   91. The system of any one of claims 88 to 90, wherein two or more    individual microfluorometers of the plurality further comprise or    share at least two excitation radiation sources.

-   92. The system of any one of claims 88 to 91, wherein the objectives    of the individual microfluorometers of the plurality have a    numerical aperture between 0.2 and 0.5.

-   93. The system of any one of claims 75 to 92, wherein the    microfluorometers of the plurality are configured to acquire images    at a resolution sufficient to distinguish features that are less    than 50 microns apart.

-   94. The system of any one of claims 82 to 93, wherein the    microfluorometers of the plurality are configured to have a    depth-of-field that is less than the separation distance between the    first and second interior surfaces of the flow cell.

-   95. The system of any one of claims 82 to 94, wherein the first    subset of the plurality of microfluorometers is configured to    acquire wide-field images at a first fluorescence emission    wavelength and the second subset of the plurality of    microfluorometers is configured to acquire wide field images at a    second fluorescence emission wavelength.

-   96. A method of sequencing a nucleic acid molecule, the method    comprising:    -   a) providing a surface; wherein the surface comprises:        -   i) a substrate;        -   ii) at least one hydrophilic polymer coating layer;        -   iii) a plurality of oligonucleotide molecules attached to at            least one hydrophilic polymer coating layer; and        -   iv) at least one discrete region of said surface that            comprises a plurality of clonally-amplified, sample nucleic            acid molecules immobilized to said plurality of attached            oligonucleotide molecules, wherein said plurality of            immobilized clonally amplified sample nucleic acid molecules            are present at distance less than λ/(2*NA), wherein a, is            the center wavelength of an excitation energy source and NA            is the numerical aperture of an imaging system.    -   b) applying a stochastic photo-switching chemistry to said        plurality of clonally amplified sample nucleic acid molecules at        the same time to cause said plurality of clonally amplified        sample nucleic acid molecules to fluoresce in on and off events        in up to four different colors by stochastic photo-switching;        and    -   c) detecting said on and off events in a color channel for each        color in real-time as the on and off events are occurring for        said plurality of clonally amplified sample nucleic acid        molecules to determine an identify of a nucleotide of said        clonally amplified sample nucleic acid molecule.

-   97. The method of claim 96, wherein concentrations of reagents for    said stochastic photo switching are sufficient such that the    probability that an on event for a given nucleotide for a given    clonally amplified sample nucleic acid molecule of said plurality of    clonally amplified sample nucleic acid molecules will occur at the    same time as an on event for a given nucleotide of a clonally    amplified sample nucleic acid molecule adjacent to said given    clonally amplified sample nucleic acid molecule is less than about    0.5%.

-   98. The method of claim 96, further comprising, controlling a rate    at which said on and off events occur to control a probability that    an on event for a given nucleotide for a given clonally amplified    sample nucleic acid molecule will occur at the same time as an on    event for a nucleotide of a clonally amplified sample nucleic acid    molecule adjacent to said given clonally amplified sample nucleic    acid molecule.

-   99. The method of claim 98, wherein controlling said rate at which    said on and off events occur comprises adjusting concentrations of    nucleotides and enzymes in said stochastic photo-switching    chemistry.

-   100. The method of claim 96, further comprising, determining whether    an illumination intensity of a detection event in a color channel is    greater than a predetermined threshold.

-   101. The method of claim 96, further comprising, determining whether    a spot size of a detection event in a color channel is greater than    a predetermined threshold.

-   102. The method of claim 96, wherein said at least one hydrophilic    polymer coating layer comprises PEG.

-   103. The method of claim 96, wherein detecting comprises acquiring    an image of said surface, wherein said image exhibits a contrast to    noise ratio (CNR) of at least 40.

-   104. The method of claim 96, wherein detecting comprises acquiring    an image of said surface, wherein said image exhibits a contrast to    noise ratio (CNR) of at least 60.

-   105. The method of claim 96, wherein said substrate comprises glass.

-   106. The method of claim 96, wherein said substrate comprises    plastic.

-   107. The method of claim 96, wherein said surface is positioned on    the interior of a flow channel.

-   108. The method of claim 96, wherein said at least one hydrophilic    polymer layer comprises a branched hydrophilic polymer having at    least 8 branches.

-   109. The method of claim 96, wherein a background fluorescence    intensity measured at a region of said surface that is    laterally-displaced from said at least one discrete region is no    more than 2× of the intensity measured at said at least one discrete    region prior to said clonal amplification.

-   110. The method of claim 96, wherein said sample nucleic acid    molecules comprise single-stranded multimeric nucleic acid molecules    comprising repeats of a regularly occurring monomer unit.

-   111. The method of claim 110, wherein said single-stranded    multimeric nucleic acid molecules are at least 10 kb in length.

-   112. The method of claim 110, further comprising double-stranded    monomeric copies of the regularly occurring monomer unit.

-   113. The method of claim 96, wherein said surface comprises a first    layer comprising a monolayer of polymer molecules tethered to a    surface of said substrate; a second layer comprising polymer    molecules tethered to said polymer molecules of said first layer;    and a third layer comprising polymer molecules tethered to said    polymer molecules of said second layer, wherein at least on layer    comprises branched polymer molecules.

-   114. The method of claim 113, wherein said third layer further    comprises oligonucleotides tethered to said polymer molecules of    said third layer.

-   115. The method of claim 114, wherein said oligonucleotides tethered    to said polymer molecules of said third layer are distributed at a    plurality of depths throughout said third layer.

-   116. The method of claim 113, further comprising a fourth layer    comprising branched polymer molecules tethered to said polymer    molecules of said third layer, and a fifth layer comprising polymer    molecules tethered to said branched polymer molecules of said fourth    layer.

-   117. The method of claim 116, wherein said polymer molecules of said    fifth layer further comprise oligonucleotides tethered to said    polymer molecules of said fifth layer.

-   118. The method of claim 117, wherein said oligonucleotides tethered    to said polymer molecules of said fifth layer are distributed at a    plurality of depths throughout said fifth layer.

-   119. The method of claim 96, wherein said at least one hydrophilic    polymer coating layer, comprises a molecule selected from the group    consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA),    poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic    acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM),    poly(methyl methacrylate) (PMA), poly(-hydroxylethyl methacrylate)    (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate)    (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside,    streptavidin, and dextran.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in any combination in practicing the invention.It is intended that the following claims define the scope of theinvention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method of processing a nucleic acid molecule,said method comprising: (a) providing a flow cell comprising (i) a firstsurface comprising a first plurality of primed nucleic acid sequencescoupled thereto, and (ii) a second surface comprising a second pluralityof primed nucleic acid sequences coupled thereto, wherein said firstplurality of primed nucleic acid sequences and said second plurality ofprimed nucleic acid sequences are present at distance less than adiffraction limit of light comprising λ/(2*NA), wherein said λ is acenter wavelength of an excitation energy source and said NA is anumerical aperture of an imaging system for imaging said first surfaceand said second surface, and wherein said second surface isaxially-displaced from said first surface; (b) contacting said firstplurality of primed nucleic acid sequences and said second plurality ofprimed nucleic acid sequences with (i) a polymerizing enzyme and (ii) aplurality of nucleotide moieties comprising a detectable label underconditions sufficient to cause a subset of said first plurality ofprimed nucleic acid sequences and said second plurality of primednucleic acid sequences to undergo a nucleotide binding reaction therebyemitting an optical signal from said detectable label when a nucleotideof said subset of said first plurality of primed nucleic acid sequencesand said second plurality of primed nucleic acid sequences iscomplementary to a nucleotide moiety of said plurality of nucleotidemoieties; and (c) imaging, below said diffraction limit of light, saidfirst surface and said second surface with said imaging system todetermine an identity of said nucleotide of said subset of said firstplurality of primed nucleic acid sequences and said second plurality ofsaid primed nucleic acid sequences.
 2. The method of claim 1, whereincontacting in (b) further comprises applying a stochasticphoto-switching chemistry to said first plurality of primed nucleic acidsequences and said second plurality of primed nucleic acid sequencessuch that said optical signal emits in on and off events, wherein saidon and off events correspond to a nucleotide binding event of saidnucleotide binding reaction.
 3. The method of claim 2, wherein saidimaging in (c) comprises detecting said one and off events in real-timefor said subset of said first plurality of nucleic acid sequences andsaid second plurality of nucleic acid sequences.
 4. The method of claim1, wherein said optical signal is a fluorescent signal.
 5. The method ofclaim 3, wherein said on and off events occur in up to four differentcolors by stochastic photo switching.
 6. The method of claim 5, whereinsaid imaging is performed in a color channel of said flow cell for eachcolor of said up to four different colors.
 7. The method of claim 3,wherein the probability that an on event for a first nucleotide of afirst primed nucleic acid sequence of said first plurality of primednucleic acid sequences will occur at the same time as an on even for asecond nucleotide of a second primed nucleic acid sequence of said firstplurality of primed nucleic acid sequences is less than about 0.5%, when(i) said first primed nucleic acid sequence is different than saidsecond primed nucleic acid sequence, and (ii) a first optical signalemitted from a first nucleotide binding reaction for said first primednucleic acid sequence is the same as a second optical signal emittedfrom a second nucleotide binding reaction for said second primed nucleicacid sequence.
 8. The method of claim 3, further comprising controllinga rate at which said on and off events occur to control a probabilitythat an on event for a first nucleotide of a first primed nucleic acidsequence of said first plurality of primed nucleic acid sequences willoccur at the same time as an on event for a second nucleotide of asecond primed nucleic acid sequence of said first plurality of primednucleic acid sequences when said first primed nucleic acid sequence isadjacent to said second primed nucleic acid sequence.
 9. The method ofclaim 8, wherein controlling said rate at which said on and off eventsoccur comprises adjusting concentrations of reagents in said stochasticphoto-switching chemistry that are applied to said first plurality ofprimed nucleic acid sequences and said second plurality of primednucleic acid sequences.
 10. The method of claim 1, wherein said firstsurface, said second surface, or any combination thereof furthercomprises a hydrophilic polymer layer.
 11. The method of claim 10,wherein said hydrophilic polymer layer comprises a branched hydrophilicpolymer having at least 8 branches.
 12. The method of claim 10, whereinsaid hydrophilic polymer layer comprises a molecule selected from thegroup consisting of polyethylene glycol (PEG), poly(vinyl alcohol)(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylicacid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM),poly(methyl methacrylate) (PMA), poly(-hydroxyl ethyl methacrylate)(PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate)(POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside,streptavidin, and dextran.
 13. The method of claim 2, further comprisingcomparing an illumination intensity of an on event of said on and offevents with a predetermined threshold illumination intensity to resolvewhen more than one of said optical signal is emitted from more than oneof said detectable label of the same type.
 14. The method of claim 1,wherein two or more primed nucleic acid sequences of said firstplurality of primed nucleic acid sequences or said second plurality ofprimed nucleic acid sequences are comprised in a single-strandedmultimeric nucleic acid molecule.
 15. The method of claim 14, whereinsaid single-stranded multimeric nucleic acid molecule is at least 10kilobases (kb) in length.
 16. The method of claim 1, wherein contactingin (b) further comprises performing a primer extension reaction of saidsubset of said first plurality of primed nucleic acid sequences or saidsecond plurality of primed nucleic acid sequences.
 17. The method ofclaim 1, wherein said nucleotide binding reaction comprises a nucleotideincorporation reaction.
 18. The method of claim 1, wherein a nucleotidemoiety of said plurality of nucleotide moieties is conjugated to apolymer core to form a conjugated polymer-nucleotide.
 19. The method ofclaim 18, wherein said contacting in (b) comprises contacting said firstplurality of primed nucleic acid sequences and said second plurality ofprimed nucleic acid sequences with said conjugated polymer-nucleotide toform a transient binding complex between (i) at least one primed nucleicacid sequence of said first plurality of primed nucleic acid sequencesor said second plurality of primed nucleic acid sequences and (ii) anucleotide moiety of said conjugated polymer-nucleotide, when saidnucleotide moiety is complementary to a nucleotide of said at least oneprimed nucleic acid sequence.
 20. The method of claim 19, whereinimaging in (c) comprises detecting said on and off events in real-timefor said transient binding complex.
 21. The method of claim 1, wherein:(i) performing said nucleotide binding reaction comprises performing aprimer extension reaction of said subset of said first plurality ofprimed nucleic acid sequences or said second plurality of primed nucleicacid sequences; (ii) contacting in (b) further comprises applying astochastic photo-switching chemistry to said first plurality of primednucleic acid sequences and said second plurality of primed nucleic acidsequences such that said optical signal emits in on and off events in upto four different colors by stochastic photo switching, wherein said onand off events correspond to a nucleotide incorporation event of saidprimer extension reaction, and wherein a rate of said on and off eventsis controlled by adjusting concentrations of reagents in said stochasticphoto-switching chemistry that are applied to said first plurality ofprimed nucleic acid sequences and said second plurality of primednucleic acid sequences; (iii) said imaging in (c) is performed in acolor channel of said flow cell for each color of said up to fourdifferent colors, and comprises detecting said one and off events inreal-time for said subset of said first plurality of nucleic acidsequences and said second plurality of nucleic acid sequences; and (iv)the probability that an on event for a first nucleotide of a firstprimed nucleic acid sequence of said subset of said first plurality ofprimed nucleic acid sequences will occur at the same time as an on evenfor a second nucleotide of a second primed nucleic acid sequence of saidfirst plurality of primed nucleic acid sequences is less than about0.5%, when (i) said first primed nucleic acid sequence is different thansaid second primed nucleic acid sequence, and (ii) a first opticalsignal emitted from a first nucleotide binding reaction for said firstprimed nucleic acid sequence is the same as a second optical signalemitted from a second nucleotide binding reaction for said second primednucleic acid sequence.